Protective Effect Of Gloydius Brevicaudus Venom Plasminogen Activator Against Cerebral Ischemia/reperfusion Injury In Mice

Healthy male Kunming mice as the research object, adopting suture method toestablish the model of cerebral ischemia reperfusion, Gloydius brevicaudus venomplasminogen （GBV-PA） preconditioning on focal cerebral ischemia reperfusion injury.Evaluation of intervention of infarct volume，neruological deficit scores、SOD enzymeactivity、MDA expression, and activation of GBV-PA on PI3K/AKT signalingpathway in ischemia reperfusion injury after cerebral protective mechanism isdiscussed.Ⅰ Preparation of Kunming mice models of focal cerebral ischemia-reperfusioninjury by suture methodAccording to Longa and koizumi to set up MCAO with suture method,16healthy male Kunming mice, weighing25-28g, were randomly divided into shamoperation group and ischemia reperfusion group, with8rats in each group. Ischemiareperfusion model group with suture at the right middle cerebral arterial blood for2h,and the pulling thread to CCA（Common carotid artery） to MCA（Middle cerebralartery） to restore blood supply, measuring the volume of cerebral infarction in24hafter neurological deficit by Longa score, Results in ischemia reperfusion modelgroup mice had neurological deficit symptoms, manifestation of different degree ofcircle, writhing（forelimb and hindlimb walking can touch）,and even coma.Ischemiareperfusion model group compared with the sham operation group, Longa scoreincreased significantly（P<0.01）, there were significant differences, TTC stainingshowed focal infarction, model successfully reproduced.Ⅱ The purification of Gloydius brevicaudus venom Plasminogen activator andits thrombolytic effect on experimental cerebral thrombosis in miceAccording to the separation and purification process of our laboratory, theprocess is as follows: GBV→Benzamidine Sepharose^TM4FF→SuperdexG30→GBV-PA.48healthy male Kunming mice, weighing25-28g, were randomly divided intosham operation group, ischemia reperfusion model group, GBV-PA480，320和 160μg/kg groups and u-PA1AU/kg, a total of6groups, with8rats in each group.except the sham operation group, all mice were used in the suture method ischemia2h/reperfusion24h. Ischemia and2h/reperfusion after24h by Longa score method,neurological impairment score; TTC staining method for the determination of mousecerebral infarction volume ratio. Results GBV-PA high, low dose group can reduce thevolume of cerebral infarction, GBV-PA high, low dose group of infarction volumewas13.7±2.3,13.8±1.2,20.9±2（%） and ischemia reperfusion model group31±1.4（%） compared to the infarct volume was significant （P<0.05）, and GBV-PA canreduce mice the volume of cerebral infarction. GBV-PA high, low dose group weresignificantly improved and signs and symptoms of neurological deficits in ischemiareperfusion in mice, reduce the neurological deficit score, GBV-PA high, medium,low dose group of neurological deficit scores were1.37±0.92,1.25±0.70,1.63±0.92and ischemia reperfusion model group2.50±0.53there is significant difference（P<0.01or P<0.05）, and GBV-PA can reduce the neurological deficit symptoms inmice.Ⅲ Experimental study of Gloydius brevicaudus venom Plasminogen activator（GBV-PA） protective effect on mice with focal cerebral ischemia reperfusioninjury.Using48clean and healthy Kunming mice, weighing25-28g, were randomlydivided into sham operation group, ischemia reperfusion group the medicineintervention group（GBV-PA high、medium and low dose group and u-PA positivecontrol group）, with8rats in each group. Ischemia120min/reperfusion24h rat modelof MCAO prepared. Ischemia120min/reperfusion24h using the Longa score ofneurological impairment score, TTC staining was used to determine the volume ofcerebral infarction of rats. The experimental results show that the mouse neuralbehaviors of GBV-PA pretreatment were significantly improved. The mouse neuralbehaviors of GBV-PA medium dose group is lower than that of model group（P<0.05）.The cerebral infarction volume percentage of GBV-PA medium dose group wassignificantly smaller than the low dose group and u-PA group. Ⅳ The mechanism of the protective effect of Gloydius brevicaudus venomplasminogen activator （GBV-PA） on mice with focal cerebral ischemiareperfusion injury.32healthy male Kunming mice, weighing25-28g, were randomly divided intosham operation group, ischemia reperfusion group, the GBV-PA320μg/kg group andu-PA1AU/kg positive control group, with8rats in each group. Animal model:reperfusion24h preparation of rat MCAO model. Sham operation group did insertthread. After24h detection of SOD activity and MDA content of brain. Another20mice, were randomly divided into sham operation group, ischemia reperfusion group,the GBV-PA （320μg/kg） group and u-PA （1AU/kg） positive control group, with5ratsin each group. Using the side of infarction penumbra based site, for the detection ofbrain tissue p-Akt expression level. The experimental results show that, comparedwith the model group, GBV-PA（320μg/kg） group and u-PA（1AU/kg）could increaseSOD activity in brain tissue（P<0.05）, and also decrease MDA content in braintissue（P<0.05）. Pretreatment of GBV-PA could increase the expression of p-akt inbrain tissues. GBV-PA pretreatment of mice with focal cerebral ischemia reperfusioninjury, PI3K/AKT signaling pathways involved in mediating the protective effect ofGBV-PA preconditioning.