| Orphanin FQ receptor (opioid receotor-like1 receptor,ORL1), a newly discovered endogenous opioid peptide receptor in recent years widely distributed in central and peripheral organizations, to participate in a variety of physiological activities of the body. Nociceptin (orphanin-FQ, OFQ) is found only nociceptin receptor endogenous legend and plays a very important role in the body .Literature shows that the nociceptin (OFQ) increased significantly after ischemia-reperfusion in the brain, The increasing of OFQ affect the local neuron's excitability through the its receptor ORL1, and influence the neuron transmitter release of local neuron and blood distribution of the parts of the brain. On this evidence some scholars speculate that the OFQ/ORL1 system may play an important role in ischemic brain injury. In this study, we investigated the effect of the different does and different injection time on neuronal ischemia-reperfusion injury, through administrating Nphe to the lateral cerebral ventricle in the focal cerebral ischemia rat model. Our research provided the very important evidence to further understand the effect of OFQ/ORL1 system on neuronal ischemia-reperfusion injury.Part one The protective effect of different doses of Nphe pretreatment on focal cerebral ischemia neuronal injury in ratsAims: To investigate the protective effect of different doses of Nphe pretreatment on focal cerebral ischemia neuronal injury in rats.Methods: 78 male SD rats weight of 280~320g were randomly divided into sham operation group(S group), the control group(C group)and four different doses of the treatment groups (T0.03,T0.3,T3,T30), 13 rats in each group, Each groups were randomly divided in two parts, of which 10 rats suffered behavioral score(NDS score) and the cerebral volume measurements in the 24h after ischemia-reperfusion The other 3 rats suffered execution of decapitation and the caspase-3 was detected by the Western-Blot in the hippocampus and striatum. Control group and Treatment groups were suffered the suture model of middle cerebral artery occlusion (MCAO), resuming the reperfusion after 2 hours ischemia. Sham group was suffered the same operation with control group but not placed the nylon line. Treatment groups respectively,45 minutes before the surgery,were intracerebroventricular administrated the orphanin FQ receptor antagonist Nphe 0.03 nmol, 0.3nmol, 3 nmol and 30 nmol, the volume of 10 ul, At the same time, Sham group and C group were intracerebroventricular administrated the same volume of physiology saline .Results: NDS score the study of neurological score showed: Score of S group had no difference with the normal animals. Scores of control and T0.03 groups decreased significantly, but there is no significant difference between the two groups. Scores of T0.3, T3 and T30 groups significantly improved compared with the control group(P<0.05). The effect of T3 and T30 groups were more significant than T0.3. There is significant difference between the two groups and T0.3 group (P<0.05).Cerebral Infarction volume The sham group had no infarction. Control group (40.23±7.25%) and T0.03 group (39.88±5.55%) were higher than T0.3 group (28.47±5.89%), T3 group (21.08±5.71%) and T30 group (19.98±5.27%) (P<0.05), Which shows 0.3nmol, 3nmol and 30nmol of orphaning FQ receptor antagonist can reduce ischemia-reperfusion injury of neurons, and 3nmol and 30nmol antagonist have more pronounced effect (P<0.05), but inter-groups have no significant difference(P >0.05).The result of infarction volume is same with the NDS.Western-blot The result s show that expression of Capase-3 in control and T0.03 groups increased significantly in hippocampus and striatum tissue, compared with the other groups after 24 hours ischemia- reperfusion. In T0.3, T3 and T30 groups, Although Caspase-3 expression was inhibited significantly, it is still significantly higher than S group. Part two The Nphe's protective effect on focal cerebral ischemia neuronal injury at different injection timeAims: To investigate the Nphe's protective effect on focal cerebral ischemia neuronal injury at different injection time.Methods: Forty-five male SD rats weight of 280-320g was randomly divided into control group (C group) and four treatment groups (T groups). At the four times points , the ischemic 1h (T1h group), the ischemic reperfusion 1h (T1H group), the reperfusion 4h (T4H group),and the reperfusion 12h(T12H group),The rats were given intracerebroventricular the ORL1 antagonist (Nphe) 3nmol, volume of 10ul. C and T groups were suffered the same suture model of middle cerebral artery occlusion (MCAO), and resume the reperfusion after two hours ischemia. Each groups were randomly divided in two parts, of which 6 rats suffered behavioral score (NDS score) and the cerebral volume measurements in the 24h after ischemia-reperfusion .The other 3 rats suffered execution of decapitation and the caspase-3 was detected by the Western-Blot in the striatum. Results: NDS score Compared with the control group, behavioral score of T1h, T1H groups were obvious improved. There is significantly difference between the groups (P<0.05).The score of T4H group is higher than control group, but inter-groups difference have no statistical significance(P >0.05). T12H group showed no obvious difference with control group.Cerebral Infarction volume The infarction volume of T1h group (26.32±6.75%) and T1H group (30.38±6.06%) are significantly smaller than C group (39.92± 6.25%) (P<0.05). T4H group(36.00±9.60%)and T12H group (39.79±8.40%)of the infarct volume has been reduced, compared with control group, but there is no difference between the groups (P >0.05).Western-blot The results show that the expression of Caspase-3 protein in T1h, T1H groups was significantly inhibited compared with the C group in the 24h after ischemia-reperfusion. T1h, T1H groups are significantly lower than T4H, T12H groups.Conclusions1. A certain does of Nphe pretreatment can significantly relieve the ischemia-reperfusion injury of neurons. And have a post-ischemic neuronal protection. The protective effect relatively depends on the dose of Nphe.2. The protective effect on ischemia neurons of Nphe depend on the time of administration after ischemia, the preventive effect is better than treatment effect when administrated the effective dose of Nphe.3. Nphe can obviously inhibit apoptosis protein synthesis after ischemia-reperfusion, shows the protective effect on neurons of Nphe can be caused by inhibiting apoptosis protein synthesis...
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