The Function Of Bradykinin Receptors In Cerebral Ischemia-reperfusion

Cerebral arterial thrombosis is one of the most common diseases that cause unnatural death and disability. Kallikrein-kinin system as an agreed inflammation regulatory system plays an important role in the pathophysiologic process after cerebral ischemia. However, reseaches from recent years implied that this system may produce completely different effects during various stages of cerebral ischemia. Bradykinin as the terminal effective factor of this system can influence the cell apoptotic process after cerebral ischemia by activating its B1 and B2 receptors. In the present study, we first observed the time course of each component of KKS and cellular distribution of bradykinin receptors in ischemic brain tissue. Then specific bradykinin receptor antagonists were used at different time points after brain ischemic damage to further explore the function of KKS in various phases of cerebral ischemia. In order to avoid the influence of the complicated environment in vivo we cultured neurons which were then deprived of oxygen and glucose(OGD) followed by oxygen-glucose re-supply. Specific bradykinin receptor agonists and antagonists were used to intervene these neurons, then neuronal apoptosis was observed. Through this study, we wish to further understand the role of KKS on various stages of cerebral ischemia and look forward to providing a promising target for the treatment of cerebral infarction.PartⅠThe expression of Kallikrein-Kinin System at different time points after cerebral ischemiaTo study the time course of each components of KKS after cerebral ischemia is necessary for the exploration of its function in various phases of this pathologic process.Rats were rendered to undergo 90 minutes of the left middle cerebral artery occlusion(MCAO) by the intraluminal thread technique before reperfusion. We tested the enzyme activity of tissue kallikrein(TK), the concentration of bradykinin(BK), the expression and distribution of bradykinin B1 and B2 receptors in the ischemic brain tissue before ischemia and at the different time points after reperfusion. Brain tissue of the left hemisphere 1mm before and after bregma (equal to the ischemic core) was taken to be examined by bio-enzymatic methods and ELISA for the determination of the enzyme activity of tissue kallikrein and the concentration of bradykinin respectively. The time points were as follows: before ischemia, 2h, 4h, 6h, 12h, 24h and 48h after reperfusion. RT-PCR and Western-blot were employed to detect the expression of mRNA and protein levels of bradykinin receptors before ischemia and at the 3rd, 6th, 12th, 24th, 48th, 72th hour and the 4th, 7th day after reperfusion. According to the results of Western-blot, the distribution of bradykinin B1 and B2 receptors on neurons and astrocytes were observed by double fluorescent staining at the peak expressing time points of B1 and B2 receptors, that is, 12h and 72h after reperfusion respectively.Results showed that: 1. Ischemic damage to the brain resulted in the up-regulation of the enzyme activity of tissue kallikrein and the concentration of bradykinin who had the same time course with the peak time points at the 2nd hour after reperfusion. 2. The mRNA and protein level of B1 receptor were rapidly up-regulated after ischemia, peaked at the 12th hour after reperfusion and returned to baseline at the 4th day after reperfusion with a snap decline. However, the mRNA and protein level of B2 receptor gradually increased to peak at the 48～72nd hour after reperfusion. This high level maintained for at least 7 days with a slow decline. 3. After cerebral ischemia, B1 receptor was mainly located on the reactive astrocytes. But B2 receptor was predominantly distributed on cortical neurons in ischemic marginal zone.The above results implied: The time course and distribution of B1 and B2 receptors were totally different after ischemic damage which could explain the different function of KKS on various stages after cerebral ischemia.PartⅡThe function of bradykinin receptors at different stages after cerebral ischemiaWe observed that the time course and distribution of B1 and B2 receptors were totally different after ischemic damage. To further explore the functions of KKS in cerebral infarction especially the bradykinin B1 and B2 receptors, Lys-(des-Arg~9-Leu~8)-BK and bradyzide as the specific antagonist of B1 receptor and B2 receptor respectively were employed for the treatment of cerebral ischemia models. The influence of these two receptors on ischemic prognosis was anticipated to be revealed. The same MCAO modals as in partⅠwere used in this part.The animal models were divided into immediate intervention group and intervention after 24h group. In immediate intervention group, bradykinin B1 or B2 receptor antagonists were administrated immediately through vena caudalis after reperfusion, then the animals were sacrificed 24h after reperfusion. In the intervention after 24h group, animals were not treated with the above drugs until the 24th hour after reperfusion. After 7 days intervention once a day the rats were executed at 7th day after reperfusion. As to the means of intervention each of the above group was subdivided into four subgroups as follows: sham-operated group, saline intervened group (with saline 2ml/kg), B1 receptor antagonist group (300μg/kg) and B2 receptor antagonist group (0.75 nmol/kg).Neurological function was appraised by NSS, infarct volume was determined by TTC stain, permeability of BBB was measured by evans blue extravasation, ultrastructure of BBB was observed by transmission electron microscope, the concentrations of inflammatory factors in ischemic tissue were measured by ELISA, the apoptosis in ischemic core and penumbra were detected by TUNEL, and the expression of B1 and B2 receptor were inspected by Western-blot.The results showed that: 1. In the immediate intervention group the antagonists of bradykinin B1 and B2 can both dramatically improve the neurological function, decrease the infarct volume and permeability of BBB, maintain the integrity of BBB, suppress the secretion of inflammatory factors and relieve the cell apoptosis with a more obvious effect for B2 receptor antagonist. 2. However, in the intervention after 24h group the two antagonists have distinguished disparation.. B1 receptor antagonist Lys-(des-Arg~9-Leu~8)-BK can reduce the cell apoptosis, decrease the infarct volume and ameliorate the neurological fuction. B2 receptor antagonist bradyzide can enhance the cell apoptosis in ischemic zone, increase the infarct volume, aggravate the neurologic injury induced by ischemia. 3. Furthermore, B2 receptor antagonist has an obviously inhibitory effects on the expression of B1 receptor. But the B1 receptor antagonist has no significant impact on the expression of B2 receptor.The results implied: 1. At the earlier stage after reperfusion, both bradykinin B1 and B2 receptors can aggravate the tissue damage of cerebral ischemia. 2. At the later stage after reperfusion bradykinin B2 receptor can inhibit the cell apoptosis induced by ischemic damage. 3. The activation of B2 receptor can increase the expression of B1 receptor. PartⅢThe effects of bradykinin receptors on the apoptosis of neurons undergoing oxygen and glucose deprivationWe observed that bradykinin B1 and B2 receptors have conspicuously different effect on cell apoptosis induced by ischemia. In order to avoid the influence of the complicated environment in vivo. In this section we cultured neurons which were then deprived of oxygen and glucose (OGD) followed by oxygen-glucose re-supply. The neurons were then intervened with specific bradykinin receptor agonists and antagonists to further illuminate the effects of bradykinin receptors on the apoptosis of neurons.Primary rat cortical neurons in good condition were used for the successive experiment after cultured in vitro for 7-10 days. After endured 1.5h of oxygen and glucose deprivation followed by oxygen-glucose re-supply, cell survival rate were measured by MTT to determine the time course of oxygen-glucose re-supply. The expression of bradykinin B1 and B2 receptors on cultured neurons were detected by double fluorensent stain. The cultured neurons were intervened with B1 receptor agonist (des-Arg~9-BK, 1μmol/L), B2 receptor agonist (BK, 1μmol/L), B1 receptor antagonist [Lys-(des-Arg~9-Leu~8)-BK, 100nmol/L] and B2 receptor antagonist (HOE140, 100nmol/L) 24h before oxygen and glucose deprivation. Cell apoptosis of the neurons were inspected by TUNEL 4 hours after oxygen-glucose re-supply.The results showed that: 1. Both bradykinin B1 and B2 receptors are expressed on cultured neurons. The expression of these two receptors could up-regulated after oxygen glucose deprivation. 2. The cell survival rate declined 50% after OGD 1.5h followed by oxygen-glucose re-supply 4h. 3. B1 receptor agonist and B2 receptor antagonist could prominently enhance the neuron apoptosis induced by OGD. B1 receptor antagonist and B2 receptor agonist could markedly suppress the neuron apoptosis.The results implied that: 1. Bradykinin B1 receptor can increase neuron apoptosis induced by OGD. 2. Bradykinin B2 receptor can inhibit the neuron apoptosis. Conclusions1. Bradykinin B1 receptor mainly located on the reactive astrocytes after cerebral ischemia was up-regulated rapidly after reperfusion with the function of aggravate the ischemia induced brain damage by increasing the permeability of BBB and the secretion of inflammatory factors. The activation of B1 receptor distributed on neurons can enhance neuronal apoptosis induced by OGD.2. Braykinin B2 receptor mainly distributed on neurons in the marginal zone of ischemia was up-regulated gradually after reperfusion. It has an anti-apoptotic function although at the early stage of cerebral ischemia B2 receptor can aggravate brain damage by increasing the expression of B1 receptor.