The Function And Mechanism Of Tissue Kallikrein To The Rat Model Of Cerebral Ischemic-reperfusion

Stroke is a common disease in aged people with high mortality and disability rate.The present situation in treatment of cerebral infarction is not satisfying.Thrombolysis is the only effective treatment that was generally adopted to acute cerebral infarction.But the time window of thrombolysis is short.There is a potential risk of secondary cerebral hemorrhage.Therefore,the clinical application of thrombolysis limited.It was the brain protection that become a hot topic appeared in clinical.The kallikrein-kinin system(KKS) is generally distributed over body's organism and produce several effects of pathology and physiology such as the blood pressure adjustment,the stabilization of environment in vivo,the growth and infiltration of tumors,inflammatory reaction,the hemal anagenesis and so on.The KKS is also generally distributed over central nervous system.The KKS play an important regulative effect in several pathological course such as cerebrovascular disease and the degeneration,inflammation,tumor in central nervous system.The effect of TK to cerebral infarction is a research hotspot and exists dispute.Some researches show that in the beginning of rat cerebral ischemia,application of TK can aggravate inflammatory reaction in the cerebral ischemic organism and can exacerbate the cerebral ischemia.Some specialists consider that after cerebral ischemia,delayed utilization TK (utilization after ischemia 8 hours) can ease cerebral ischemia and furthermore benefit the recovery of the cerebral ischemia.Moreover this kind protection through Bradykinin B2 receptor produces a marked effect.Blockage of the Bradykinin B2 receptor,this kind of protection was removed.Our research mainly explores the delayed application of TK(utilization after reperfusion 8 hours) dose have protection;the mechanism of action to the organism of cerebral ischemia;the influence to Bradykinin and its receptor in ischemic region.Through this research,we wish to identify the effect and mechanism of TK to cerebral ischemia.We wish to get new treatment target to cerebral infarction.PartⅠThe effect of tissue kallikrein to the cerebral ischemia- reperfusion ratsObjective:To explore the effect of delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion.Method:All rats were randomly divided into three groups.Group A was sham operation; group B was control group of physiological saline;group C was treatment group of TK. The manufacture of cerebral ischemic-reperfusion rat model was induced by middle cerebral artery occlusion(MCAO).After ischemia 8 hours,the reperfusion was restored. After reperfusion 8 hours,the control group rats were intraperitoneally administrated with physiological saline at dose of 2ml·kg^-1d^-1 for three days;the treatment group rats were intraperitoneally administrated with TK at dose of 17.5PNAU·kg^-1d^-1 for three days.After three days,neurological function was appraised;infarct volume was determined.Pathology and Nissl's staining in ischemic tissue were detected.The cerebral protection of TK to cerebral ischemic-reperfusion rats were appraised by above methods.Result:The delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion can obviously reduce the loss of neurological function and the infarct volume.There were obvious statistics difference(P＜0.05) between group B and group C.The delayed application TK can also relieve the change of pathology and improve the Nissl's staining in the ischemic tissue.Conclusion:Delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion has protective effect to cerebral ischemia.PartⅡThe mechanism of tissue kallikrein to the cerebral ischemia- reperfusion ratsObjective:To explore the protective mechanism of delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion.Method:All rats were randomly divided into three groups.Group A was sham operation; group B was control group of physiological saline;group C was treatment group of TK. The preparative method and administration of experimental rats were identical with partⅠ.To explore the mechanism by the following five aspects:first,to the influence of apoptosis in the ischemic region by detecting TUNEL staining and immunohistochemistry of Caspase-3;second,to the influence of inflammatory reaction in the ischemic region by detecting immunohistochemistry of TNF-α,IL-6;third,to the influence of neurogenesis in the ischemic region by detecting immunohistochemistry of NSE;fourth,to the influence of neurogliocyte regeneration in the ischemic region by detecting immunohistochemistry of GFAP;fifth,to the influence of angiogenesis in the ischemic region by detecting immunohistochemistry of vWF.Result:The protective mechanisms of delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion were following:first,TK can relieve apoptosis in the ischemic region.TK can reduce the positive cells of TUNEL staining and Caspase-3 immunohistochemistry.There were obvious statistics difference(P＜0.05) between group B and group C.Second,TK can relieve inflammatory reaction in the ischemic region.TK can reduce the positive cells of TNF-α,IL-6 immunohistochemistry. There were obvious statistics difference(P＜0.05) between group B and group C.Third, TK can advance neurogenesis in the ischemic region.TK can increase the positive cells of NSE immunohistochemistry.There were obvious statistics difference(P＜0.05) between group B and group C.Fourth,TK can advance neurogliocyte regeneration in the ischemic region.TK can increase the positive cells of GFAP immunohistochemistry.There were obvious statistics difference(P＜0.05) between group B and group C.Fifth,TK can advance angiogenesis in the ischemic region.TK can obviously increase the positive cells of vWF immunohistochemistry.Conclusion:The protective mechanisms of delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion were following:inhibiting apoptosis and inflammatory reaction,advancing neurogenesis,neurogliocyte regeneration and angiogenesis in the ischemic region.PartⅢThe bradykinin and its receptors affected by tissue kallikrein in the cerebral ischemia- reperfusion ratsObjective:To explore the bradykinin and its receptors affected by delayed application TK (utilization after reperfusion 8 hours) in the cerebral ischemia- reperfusion rats.Method:All rats were randomly divided into three groups.Group A was sham operation; group B was control group of physiological saline;group C was treatment group of TK. The preparative method and administration of experimental rats were identical with partⅠ.The density of bradykinin in the ischemic region was detected by ELISA method. RT-PCR was employed to detect the expression of mRNA levels of bradykinin receptors. West-blot was employed to detect the expression of protein levels of bradykinin receptors.Result:Delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion can advance the density of bradykinin.There were obvious statistics difference(P＜0.05)between group B and group C.It can lightly advance the expression of mRNA levels of bradykinin B1 receptor.There were no obvious statistics difference(P＞0.05) between group B and group C.It can obviously advance the expression of mRNA levels of bradykinin B2 receptor.There were obvious statistics difference(P＜0.05) between group B and group C.Delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion can lightly advance the expression of protein levels of bradykinin B1 receptor.It can obviously advance the expression of protein levels of bradykinin B2 receptor.Conclusion:Delayed application TK(utilization after reperfusion 8 hours) to rats of cerebral ischemic-reperfusion can advance the density of bradykinin.advance,lightly advance the expression of bradykinin B1 receptor and obviously advance the expression of bradykinin B2 receptor.