| The bovine Pancreatic Typsin inhibitor is a representative inhibitor of Kunitzfamily.BPTI extracted from bovine pancreas is a serine protease inhibitor.BPTI is anonspecific and broad-spectrum serine protease inhibitor which affects known serineproteases such as trypsin, chymotrypsin, plasmin and kallikrein. It can protect bloodplatelet against bleeding, treat the patient with cruor impediment, in which situationthe hemorrhage may occur. BPTI can be used in cardiac surgery as beingextracorporal circulated to decrease the incidence of bleeding and the inflammatoryreaction caused by the surgery. But infusion of high doses of these nonmammalianinhibitor could result in immunologic reactions in patients undergoing repeat use. Sowe need find a substitute which is preferable ,small ,potent and of human sequenceorigin.The amyloid β-protein precursor contains a domain homologous to Kunitz-typeserine protease inhibitors.The sequence of KD/APPwhich is composed of 57 aminohas 43% sequence identity with that of BPTI. The tertiary fold, reactive center andmechanism of function of KD/APP is very similar to that of the Kunitz inhibitorBPTI.The KPI containing isoforms (APPIs) show ability to inhibit a variety of serineproteases such as factor XIa, epidermal growth factor (EGF)-binding protein, theγ-subunit of nerve growth factor, especially the inhibition constant Ki of KD/APP tofactor XIa is higher than BPTI. The APPIsor is an effective cell adhesion molecule,which has growth activity. They probably play a role in the happening of series ofevents that lead to tissue repair at vascular wound sites and protect neurocytes. Thepathological and physiological role of this protease inhibitor domains which concernswith abnormal metabolism of rhKD/APP are not well understood. So we need toproduce large quantity of rhKD/APP to discover and study the possible mechanism onpharmacodynamics.Increasing evidence has shown that brain injury can develop as a result ofcerebral ischemia-reperfusion due to stroke and other cardiovascular diseases.However, the mechanism on cerebral injury is not understood. The aim of this studyis to investigate the protective effect and mechanism of on rhKD/APP cerebral injuryinduced by brain ischemia/reperfusion of rats.It can provide a theory basis for itsclinical application on cerebral visculardiseases.PartI Document ReviewThe summary of typsin inhibitor of Kunitz, BPTI and human sequence origininhibitor of Kunitz, rhKD/APP.The summary of mechanism and research development of brainischemia-reperfusion.PartII The study of the neural protective effect of rhKD/APP on ischemiareperfusion injury in rat brainThe purpose is to investigate the protective effects of rhKD/APP scinate oncerebral ischemia-reperfusion injury in rats. Rats were pretreated with rhKD/APP for1h and then subjected to cerebral ischemia/reperfusion injury induced by a middlecerebral artery occlusion (MCAO). After 3h ischemia and 24h reperfusion, theneurological outcome was evaluated by the Longa's method;the infarct volume wasassessed by TTC staining and the cerebral water content was measured by dry-weightmethod. rhKD/APP (8,16 mg·kg-1) significantly reduced the cerebral infarct volumeand water content, and ameliorated the neurological deficit (P<0.05 or P<0.01). Theseresults showed that pretreatment with rhKD/APP could decrease brain injury aftercerebral ischemia/reperfusion.The study of the neural protective effect of rhKD/APP on ischemiareperfusion injury in rat brainPartI The study of effect f rhKD/APP on the activities of SOD, Na+-K+-ATPase and the content of MDARats were pretreated with rhKD/APP for 1h and then subjected to cerebralischemia/reperfusion injury induced by a middle cerebral artery occlusion . After 3hischemia and 24h reperfusion, the activities of SOD, Na+-K+-ATPase and the contentof MDA were measured in cortex of ischemic and non-ischemic hemisphere. Inmodel rats, the activities of SOD and Na+-K+-ATPase in the of ischemic hemispherewere all decreased and the concent of MDA was significantly increased (P<0.01) ascompared with the sham-operated group. After treated with the rhKD/APP (8,16mg·kg-1), the activities of SOD, Na+-K+-ATPase were significantly increased, whilethe content of MDA were obviously decreased (P<0.05 or P<0.01). These resultsshowed that pretreatment with rhKD/APP could increase the activities of theseantioxidant enzymes on rats after cerebral ischemisa/reperfusion.PartII The study of mechanism of rhKD/APP on the balance ofinflammary and anti-inflammary in the cerebral ischemia reperfusion of ratsThis study is to investigate the effects of rhKD/APP on neutrophil migration andadhesion molecules after middle cerebral artery occlusion in rats.Rats were pretreatedwith rhKD/APP for 1h and then subjected to cerebral ischemia/reperfusion (I/R)injury induced by a middle cerebral artery occlusion.After 3h ischemia and 24hreperfusion, the serum contents of Interleukin-8 (IL-8) was determined byradioimmunossay (RIA). After 3h ischemia and 24h reperfusion, the effect of sodiumrhKD/APP on the migration of neutrophil was evaluated by measuring the activity ofMyeloperoxidase (MPO) enzyme. The expressions of adhesion molecules (ICAM-1and E-selectin) were determined by immunohistochemistry . The results showed thatrhKD/APP group (8,16 mg·kg-1): the concent of IL-8 in serum at ischemic tissuewere obviously decreased (P<0.05 or P<0.01, vs model group);After treated withrhKD/APP (8,16 mg·kg-1), the enzymatic activity of MPO and the expressions ofthese adhesion molecules were significantly reduced compared with model group(P<0.05 or P<0.01).The results have shown that rhKD/APP has the protective effecton cerebral ischemic-induced damage through inhibiting the inflammatory process.Part III The study of the effect of rhKD/APP on apoptosis of cerebralischemia reperfusion of ratsThis section was to investigate the effects of rhKD/APP on apoptosis induced bytransient focal brain ischemia in rats. Rats were pretreated with rhKD/APP for 1h andthen subjected to brain ischemia/reperfusion (I/R) injury induced by a middle cerebralartery occlusion. After 3h ischemia and 24h reperfusion, Hematoxylin-Eosin (HE)staining, in situ end-labeling of nuclear DNA fragmentation (TUNEL) were employedto determine the level of apoptosis. The expressions of caspase-3 and Bcl-2 in thecortex were determined by immunohistochemistry and Western blot;The increasenumbers of HE-and TUNEL-positive staining cells were significantly observed at 24hafter reperfusion. The immunoreactivity was inhibited by rhKD/APP (4,8,16 mg·kg-1)(P<0.01 or P<0.05, vs vehicle-treated). After cerebral I/ R, caspase-3 was activated,whereas Bcl-2 expression was inhibited. rhKD/APP (4,8,16 mg·kg-1) markedlyinhibited the expression of caspase-3 ,up-regulated the expression of Bcl-2 anddown-regulated the expression of Bax(P<0.05, or P<0.01 vs model group). On theabove statements, we can draw a conclusion that rhKD/APP could potently inhibitcaspase-3 activation in creasing the expression of Bcl-2 and decreasingthe expressionof Bax cerebral I/R in rats. These findings on the inhibitory effect of rhKD/APP onbrain ischemic injury-induced apoptosis might have important theories basis for thetreatment on ischemic cerebrovascular diseases .Conclusions:1. rhKD/APP is first to be used in the treatment of rat's cerebral ischemiareinfusion. The results show that rhKD/APP can improve the neural fuction,reducethe infarct volume ,relieve the cerebral edma,enhance the endurance of the rat's brainto the ischemia.2. From below aspects,rhKD/APP may protect the neural injury of cerebralischemia reinfusion.This medicine has great potential ability in clinical.(1)rhKD/APPobviously increased the activities of SOD, Na+-K+-ATPase anddecreased the content MDA after cerebral ischemia/reperfusion.(2) rhKD/APP obviously inhibited the inflammatory process through decreasingthe content of IL-8 and reducing the enzymatic activity of MPO and the expressionsof the adhesion molecules (ICAM-1, E-selectin) after cerebral ischemia-reperfusion.(3)rhKD/APP inhibited the activity of caspase-3 increasing the expression ofBcl-2 and decreasing the expression of Bax cerebral I/R in rats after cerebral I/R inrats.(4) From many aspects, rhKD/APP has the protective effect of the neural fuction.These findings on the inhibitory effects of rhKD/APP on brain ischemicinjury-induced apoptosis might have important theories basis for the treatment onischemic cerebrovascular diseases.
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