Effects Of δ-opioid Receptor On The Expressions Of Synaptic Proteins In The Global Ischemic Rats

Opioid receptors are G-protein-coupled receptors involved in variety of physiological activities, such as analgesia, myocardial protection, immune response, inhibiting gastrointestinal motility, respiratory depression. Opioid receptors contain kinds of subtypes which at least three subtypes in the central nervous system:μ, κ,δ. Our experiments used [D-Ala2, D-Leu5] enkephalin(DADLE) which is Delta opioid receptor (8-opioid receptor, DOR) agonists. Recent findings indicate that, addition to analgesia, DADLE also effectively prolong storage time of transplanting organs in vitro, reduce myocardial infarct size, significantly reduce damage of neuronal cells and brain slices in vitro. However, there are several studies of ischemic injury of brain in vivo. Prior researches largely focus on ischemic preconditioning for ischemia, few studies care for the postconditioning which is more meaningful for the clinical treatment of ischemic.Our previous studies showed that DADLE which is injected into lateral ventricle of global cerebral ischemia of rats can significantly improve spatial memory function after5-9days of reperfusion. Therefore, this paper examines the expression of some proteins associated with learning and memory, in order to explore the molecular mechanisms of activated DOR on cognitive function improvement after cerebral ischemia. In addition, this paper also observed the effect of the activation of DOR on survival of neuron after cerebral ischemia.1. Build global cerebral ischemia-reperfusion model in ratsThis paper use four-vessel occlusion method (ie, blocking bilateral vertebral artery and bilateral common carotid arteries) to copy the animal models of global cerebral ischemia and reperfusion. We implant cannulae into lateral ventricle before copying the ischemia model rats in order to inject drugs which can not pass the blood brain barrier into lateral ventricular.2. The impact of DOR on neurons and astrocytes after global cerebral ischemia-reperfusionGlobal cerebral ischemia can cause neuronal loss and change astrocyte morphology of brain (especially hippocampus). The preliminary data showed that the activation of DOR can significantly improve the survival status of neurons after3days of cerebral ischemia-reperfusion. This thesis is observed the survival status of neurons after7days of cerebral ischemia-reperfusion. The results showed that the activation of DOR can reduce apoptosis of CA1neurons of hippocampus, promote activation of astrocytes, and reduce damage of astrocytes after7days of ischemia-reperfusion.3. Impact of DOR on synapsin I and its phosphorylated form after global cerebral ischemia-reperfusionSynapsin I which is located in the presynaptic membrane can regulate the release of neurotransmitter through phosphorylation and dephosphorylation. This can affect transmission of neuronal signal, thus affect the survival state of neurons and astrocytes. The chapter mainly use Western Blotting to detect the impact of DOR on synapsin I and the phosphorylated form after cerebral ischemia-reperfusion. The results showed that the expression of synapsin Ⅰ up-regulated in the I/R group after3days of ischemia-reperfusion. The expression of synapsin Ⅰ also raised in the DADLE-treated group, but the increase level was significantly larger than the I/R group. The expression levels of synapsin Ⅰ remained high in the I/R group than in the control group after7days of ischemia/reperfusion, and expression in the DADLE-treated had returned to the level in the control group. In addition, the results of Naltrindole-treated was similar with I/R group. Synaptic proteins are necessary for the repairment after cerebral ischemia-reperfusion. It can increase damage of neuron if expression is reduced. So the results suggest that the activation of DOR can accelerate the repairment of tissue in the course of ischemia/reperfusion. Second, the expression of phosphorylated synapsin Ⅰ was up-regulated in the I/R group after3days of ischemia-reperfusion, and the expression of DADLE-treated group was significantly reduced. The expression of phosphorylated synapsin Ⅰ reduced in the I/R group after7days of ischemia-reperfusion, while raised in DADLE-Treated. In addition, the results of Naltrindole-treated was similar with I/R group. Changes in the expression of phosphorylated synapsin Ⅰ can indirectly reflect transmission of neurotransmitter. The activation of DOR significantly reduced the expression of phosphorylated synapsin Ⅰ after3days of ischemia-reperfusion, thereby preventing the release of neurotransmitters, reducing excitotoxicity caused by excessive excitatory neurotransmitters. In addition, the release of neurotransmitters was recovered to establish new synaptic connections after7days of ischemia-reperfusion.4. Impact of DOR on MARCKS and its phosphorylated form after global cerebral ischemia-reperfusionMyristoylation alanine-rich C kinase substrate (MARCKS) can adjust the shape and quantity of the dendritic spine relying on phosphorylation and dephosphorylation thus affecting neuronal synaptic plasticity, and ultimately affect cognitive function. Up or down-regulation the expression level of MARCKS and its phosphorylated form is harmful for maintaining the stability of dendritic spines. Maintain the physical level is the better state. This chapter use Western Blotting to detect the impact of DOR on MARCKS and the phosphorylated form after cerebral ischemia/reperfusion. The results showed that the expression of MARCKS up-regulated in the I/R group after3days of ischemia-reperfusion. The expression of MARCKS reduced in the DADLE-Treated group. The expression of MARCKS significantly up-regulated in the I/R group after7days of ischemia-reperfusion, while the expression in the DADLE-Treated group had returned to physiological levels. The expression of phosphorylated MARCKS was down-regulated in the I/R group after3days of ischemia-reperfusion, and the expression of DADLE-Treated group was significantly reduced. The expression of phosphorylated MARCKS increased in the I/R group after7days of ischemia/reperfusion, while the expression in the DADLE-Treated group had returned physiological levels. The results of Naltrindole-treated was similar with I/R group. This suggest that the expression of MARCKS and its phosphorylated form significantly reduced after3days of ischemia-reperfusion. This may be a protective measure under pathological conditions to prevent from amplification of apoptosis signal caused by cerebral ischemia and protect neurons. Although this is unfavorable for maintaining the stability of dendritic. Expression in the DADLE-Treated group had returned to the physiological levels after7days of ischemia-reperfusion, in favour of creating new synaptic connections and reducing neuronal damages.