The Experimental Study On The Protective Effects Of Daphnetin In Brain Of Frontal Cerebral Ischemia And Reperfusion Rats

Cerebrovascular disease has threatened people healthy seriously and is one of the most common diseases. It is the first cause of death in China. The brain is our most sensitive organ to oxygen deprivation. Ischemia can lead to brain dysfunction and structural brain damage, moreover, the degree of ischemic brain damage is closely related to the duration of ischemia as well as residual blood flow. Blood flow reperfusion after cerebral ischemia cannot restore brain function, even aggravate it, which is known as cerebral ischemia reperfusion injury (CIRI). Growth-associated protein 43(GAP-43), a nervous tissue-specific phosphoric acid protein, is expressed at high levels during central nervous system (CNS) development and regeneration, which is directly bound up with neuronal growth, axons regeneration and synapse reformation. Studies have shown that GAP-43 expression increase gradually after cerebral ischemia reperfusion (CIR) and reach peak from 1w to 2w, then gradually decrease. Through activation of neuronal sprouting, GAP-43 pro-motes axonal repair, regeneration and synapse reconstruction. After nerve injury and regeneration, the level of GAP-43 in axons is increased up to 20-100 times than normal level, which is the necessary material basis for the repair, axon regeneration and functional reconstruction. Chinese extract daphnetin can inhibit neutrophil oxidative damage and exert anti-inflammatory effect. Daphnetin can expand coronary vessels, improve myocardial ischemia in the cardiovascular system and release pain, make sedation, inhibit protein kinase, regulate blood lipids and supress thrombosis in the CNS. Previouly, we found that daphnetin could improve neurological recovery by regulating glial fibrillary acidic protein in cortex and hippocampus after CIRI. This study was designed to observe effect of daphnetin on GAP-43 protein and GAP-43mRNA expression in hippocampal neurons by establishing CIRI rat model. In the end, we addressed protective mechanisms of daphnetin after CIRI from the cell, protein and molecular level.Objective:To observe the protective mechanism of daphnetin in brain of frontal cerebral ischemia and reperfusion rats.Methods:120 male Wistar rats, body weight 200-250g, are divided into three groups randomly:sham group (n=40), cerebral ischemia reperfusion group (n=40), daphnetin group (n=40). All groups are divided into 4 subgroups according to different time points:1w,2w, 4w,8w (n=10). By repeatedly clamping the bilateral common carotid artery to establish CIRI model, sham group were dissected bilateral carotid artery, no clamping. Treatment group were received intragastric administration of daphnetin from the second day after the operation till different time points. After killing the rats and removing frontal brains in different time points, we observed the hippocampus pathology and tested the expression of GAP-43 protein in hippocampus by immunohistochemistry, western-blot and the expression of GAP-43 mRNA by real-time PCR, the number of apoptotic cells in rats by TUNEL method.Results:1. Sham group were normal in hippocampal neurons under light microscope. Neurons and glial cells showed integrity structure. Ischemic neurons and glial cells were showed partly degeneration and necrosis. Neurons were round, oval, triangular, long tail-like or irregular shape, nuclear condensation. With the reperfusion time, the number of glial cells increased and the cytoplasm is rich in eosinophils red, nuclear small, dense and biased towards one side. Neuronal necrosis in treatment group were slightened compared with ischemia group.2. There was no significant difference about GAP-43 positive cells in sham group at all time points. In contrast, the expression of GAP-43 immunoreactive cells was higher in treatment group and ischemia group than in the sham group (p<0.05). It was the same as in the treatment group compared with the ischemic group (p<0.05).3. The results of Western-blot showed that there was no significant difference about GAP-43 protein in sham group at all time points. In contrast, the expression of GAP-43 protein was higher in treatment group and ischemia group than in the sham group (p<0.05). It was the same as in the treatment group compared with the ischemic group (p<0.05).4. The results of real time PCR showed that there was no significant difference about GAP-43mRNA in sham group at all time points. In contrast, the expression of GAP-43mRNA was higher in treatment group and ischemia group than in the sham group (p<0.05). It was the same as in the treatment group compared with the ischemic group (p<0.05).5. There was no significant difference about neuron apoptosis in sham group at all time points. Neuron apoptosis in hippocampus in the treatment group and ischemia group was increased compared with sham group (p<0.05). However, only a non-significant tendency of decreasing levels of neuron apoptosis was seen in treatment group compared with ischemia group (p>0.05).Conclusion:1. Daphnetin can significantly reduce neuron damage in himppocampus induced by CIRI and exert neuroprotective effect.2. Daphnetin may protect hippocampus by upregulating GAP-43 protein and the expression of GAP-43mRNA.