Effects Of Human Urinary Kininogenase On Cell Apoptosis Of Rats With Acute Cerebral Ischemia-reperfusion Injury

Background Cerebrovascular disease(CVD) is the third leading cause of death and the most common cause of disability in mankind.The General Military Cerebrovscular Research group had estimated the incidence rate,the prevalence rate, the death rate from stroke in our country by the world standard population.They are 115.61 per 100000,256.94 per 100000,81.33 per 100000,respectively,in 1990. According to WHO,because of the population accretion and aging,even if the stroke incidence rate in our country is stable,the number of stroke incidence each year will increase from 1.8 million now to 5.4 million in 2030.Ischemic Cerebrovascular Disease takes up more than 70%in CVD and is seriously harmful to huma being.The infarct of Ischemic Cerebrovascular Disease is made of the core necrotic region and peripheral ischemic penumbra.Because ischemia is complete in the core region, necrosis is the main way of cell death.But there is blood in the penumbral region in which apoptosis is the main way of cell death.The number of apoptotic neuron in ischemic penumbral may contribute to the final infarct volume.Nowadays,the focus on therapy of CVD is to recover blood in order to rescue the neuron in penumbra as soon as possible in case of apoptosis.So,the hot study on acute cerebral ischemia injury is whether drugs can inhibit the apoptosis of neuron.Apoptotic cell death is a initiatived way under physiology and pathology conditions.It is a biology process controlled by genes and factors.As we all known the Bcl-2 family protein and Caspase family protein play an important role in the apoptosis of neuron death.Bcl-2 family includes the Bcl-2,Bcl-xL,Bax,Bcl-XS,Bak, Bid,Bad,Bix etc.Among these protein,Bcl-2 is an important antiapoptotic protein,it is expressed in the neurons that be destined to survive.The most important proapoptotic protein is bax.The ratio of Bcl-2/Bax may decide the fate of neurons, death or survival.The last step of the intrinsic pathway of apoptosis is to activate Caspase-3.So,it is considered as the most important molecules involved in execution of apoptosis and lead cells to death directly.Kallikrein kinin system includes kininogenase,kininogen,kinin.According to primary structure,biochemical properties,immunological character,substrate specificity,site of synthesis,and biological function,kininogenase classifies into tissue kininogenase and plasma kininogenase.They process high molecular weight kininogen and low molecular weigh kininogen to produce Bradykinin and kallidin or vasodilatin,repectively.Because of aminopeptidase,the latter lost diaminocaproic acid and become bradykinin.The B2 receptor is constitutively expressed whereas the B1 receptor is expressed at very low levels under normal conditions and is induced by inflammation or under stress.Activation of kinin receptors modulate a broad spectrum of cellular functions including smooth muscle cell contraction or relaxation, increased vascular permeability,and cell proliferation,etc.Kinin is then cleaved by kininaseⅠorⅡ.The kallikrein-kinin system components are present in the brain,suggesting a physiological relevance in the central nervous system.KKS has a neuroprotective effect.Human urinary kininogenase is a tissue kininogenase.It comes from urinary of a healthy man.After acute cerebral ischemical reperfusion injury,HUK,through the inin B1 receptors induced by cerebral ischemia,increases intracellular Ca2+ mobilization as well as prostacyclin,and cAMP formation.Increased Ca2+ enhances endothelial nitric oxide synthase(eNOS) activity and nitric oxide(NO) production which chooses to improve the blood supply of infarct region.NO may protect against oxidative stress-induced tissue damage and stimulate the proliferation of endothelial cells and neuronal cells,then it is effective to decreased I/R-induced neurological dysfunction and cerebral infarction.Up to now,there is a lack of study about HUK on cell apoptosis of rats with acute cerebral ischemical reperfusion injury.In order to make clear if it has other mechanisms to treat ischemic cerebrovascular diseases besides vasodilatation and improving the blood supply.It also bring a example to treat cerebral ischemia for other drugs.So we design this study.PartⅠThe effect of human urinary kininogenase(HUK) on infarct sizes of rats after acute focal cerebral ischemia and reperfusion(FCIR) injuryObjective To study the effect of human urinary kininogenase(HUK) on infarct sizes after focal cerebral ischemia and reperfusion(FCIR)injury.Methods Eighteen healthy,adult,male Spraque-Dawley(SD) rats were randomly divided in to sham-operated group,ischemic-reperfusion group,and HUK-treated group according to completely random design.Each group has six rats. We established acute Middle cerebral artery occlusion(MCAO) models followed by blood reperfusion after ninty minutes of MCAO.When rats woke up,we used the scoring system described by Zea Longa et al.(1989) on a 5-point scale to evaluate neurologic impairment(0:no deficit;1:failure to extend right forepaw fully;2: circling to the right;3:falling to the right;4:no spontaneous walking).The animals scaled 1 to 3 point were enrolled into experiment(except sham-operated group).Rats were treated three hours later after blood reperfusion injury.We were done nothing to sham-operated group.I/R group and HUK group were treated with normal saline(1.0mL/kg) and human urinary kininogenase(17.5×10^-3PNAU/kg,1.0mL/kg) respectively,one time/each day.The brains of rats were removed directly after 24 h of operation and then sectioned into 2-mm coronal slices,putting the brain slices into a 2%TTC solution at 37℃and incubating for 30 minutes away from light.After that, chect out the result.TTC stains viable brain tissue red,while infracted tissue remains white.TTC-stained brain sections were photographed with a computer camera.The infarct area in each of brain slices was quantified with the use of an image processing software.The infarct volumes were calculated with formula: V=t(A1+...An)-(A1+An)t/2.t is thickness of brain.A is infarct area.Using SPSS 11.5 software for statistical treatment,the results were expressed as mean±standard deviation.Because the results of sham-operated group were zero,then they weren't analysed in order to avoiding heterogeneity of variance.Comparisons among groups were made by two independent t-test.Differences were considered significant at P＜0.05.Results No infarct volumes were found in sham-operated group.Compared HUK-treated group and I/R group,HUK significantlly reduced the infarct volume, there is a difference in statistical significance,t is 4.864,P is 0.001.Conclusion HUK can decrease the infarct volume after FCIR.Part 2 The effect of HUK on the number of apoptotic cells and the expression of Bcl-2,Bax,Caspase-3 protein of rats after acute FCIR injury.Objective To evaluate neurologic impairment scores,observe HE dyeing and measure the number of apoptotic cells and Bcl-2,Bax,Caspase-3 protein positive cells in cerebral cortex with TUNEL or immunohistochemistry after acute FCIR injury.To make sure if HUK execute protection on Ischemic Cerebrovascular Disease by inhibiting apoptosis.Methods Sixty-six healthy,adult,male Spraque-Dawley(SD) rats were divided into sham-operated group(n=6),ischemic/reperfusion group(n=30) and HUK-treated group(n=30).The latter two groups were subdivided into reperfusion 6 h(n=6),12 h(n=6),24 h(n=6),72 h(n=6),168 h(n=6) groups.Each group has six rats. To establish models and enroll rats as the same as part 1.Rats were treated three hours later after reperfusion injury.There were done nothing to sham-operated group. I/R group and HUK group were treated with normal saline(1.0mL/kg) and human urinary kininogenase(17.5×10- 3PNAU/kg,1.0mL/kg) respectively,one time/each day.Observe indexs:1.neurologic impairment scores:The rats were choosen from sham-operated group,ischemic/reperfusion72 h and 168 h group,HUK-treated 72 h and 168 h group.Using the Zea longa scoring system at time of reperfusion 6 h,12 h,24 h,48 h,72 h.2.pathobiology:After defined ischemic time(sham-operated group is 24 h after cerebral ischemia) animals were anesthetized with chloralhydrate (30ml/kg) and fixed with normal saline 250ml and 4%paraformaldehyde 300ml through apex of heart.The brains were removed from the skull.The infarcted brain were paraffin embedded.4-μm-trick paraffin sections of caudate nucleus deck were cut and used for HE staining and TUNEL for apoptosis and immunohistochemical analysis for bcl-2,Bax,Caspase-3 protein.Using SPSS 11.5 software for statistical treatment,the results were expressed as mean±standard deviation.Because the results of sham-operated group were zero,then they weren't analysed in order to avoiding heterogeneity of variance.Comparisons among groups were made by analysis of variance(ANOVA) followed by repeated measures for the neurologic impairment scores,factorial analysis for the number of apoptotic cells and Bcl-2, Bax,Caspase-3 protein positive cells.Differences were considered significant at P＜0.05.Results 1.HUK relieve the neurologic impairment of rats after FCIR,compare HUK-treat group with I/R group,the difference between them is significant at 24 h, 48 h and 72 h.2.HE stain:Compared with the I/R group,HUK-treated group decreased neuronal degeneration,ischemia,cellular necrosis and interstitial edema, especially 24 h group.3.Compare HUK-treat group with I/R group,the count of TUNEL and Bcl-2 positive cells increased,and Bax,Caspase-3 positive cells decreased except 168 h group.Conclusion HUK executed protection on FCIR injury.It decreased the number of apoptotic cells in the initial three days of FCIR injury by up-regulating Bcl-2 and down-regulating Bax,Caspase-3.