The Pharmacodynamics And Mechanisms Of Neuroprotective Effects Against Ischemic Brain Neuron Death By BDNF-PTD

BACKGROUNDThe blood-brain barrier(BBB) is mainly constituted by vascular endothelial cells and glial cells in brain tissue. Endothelial cells lack of endocytosis capsule, which limit free exchange of materials between the blood and brain tissue. Therefore, many drugs such as BDNF, a natural protein molecules in vivo, are difficult to penetrate through the blood-brain barrier. Currently, there isn't any valid, non-invasive approach which mediates BDNF through BBB to exert biological effects. How to delivery BDNF into brain is a key for its clinical application. The delivery systems for drug targeting are as follows now:1) Cerebrovascular infusion or intracerebral injection of drug.2) Viral-mediated gene-therapy.3) Liposome-encapulated drug delivery system.4) Monoclonal antibody-coupled drugs for brain targeting.However, there are many shortcomings for these methods:1) Cerebrovascular perfusion or local administration of drug in the brain has non-compliance, and the drug could only distribute in the injection area.2) Viral-mediated gene delivery into the brain is an alternative way, but it is not suit for the treatment of acute ischemic brain injury in clinic because of delayed effects. Moreover, the side effects of viral vector on human immune system and chromosome are uncertain.3) Liposome can increase the hydrophilicity of drugs. However, the brain uptake of liposomes is absolutely non-specific, which may increase the side effects of drug and cost.4) Coupled with brain-targeting monoclonal antibody, drugs could penetrate the BBB and reach the brain after intravenous administration.However, this method made the manufacture very cumbersome and expensive. How to find an effective, non-invasive delivery way to mediate drugs, such as BDNF into the brain is imminent.The protein transduction domain (PTD) discovered in Trans-Activator of Transcription(TAT) protein derived from the human immunodeficiency virus can mediate other proteins into cells by transcytosis. Protein transduction domain (PTD) are positively charged oligopeptides with less than 20 amino acids, and can transport almost all macromolecules into mammalian cells,which provides a good way to penetrate durgs through BBB into the brain.Our previous study has prepared a brain-derived neurotrophic factor-TAT/PTD fusion protein (BDNF-PTD) [9] through genetic engineering, and confirmed that the fusion protein could through the blood-brain barrier via a systemic administration. In the present study, the pharmacodynamic and the mechanism of neuroprotective effects against ischemic brain neuron death in rats by BDNF-PTD have been elucidated.OBJECTIVETo study the pharmacodynamics and neuroprotectiove function of BDNF-PTD in the animal model of cerebral ischemia, and compare the neurological deficit through the neurological deficit scores between non-treatment group and treatment group, and explore the mechanism of neuroprotective effects against brain ischemic in rats by BDNF-PTD.METHODS1) established a cerebral ischemia animal model induced by middle cerebral arterial occlusion in Sprague-Dawley ratsAdult male Sprague-Dawley rats (weight,250 to 350 g) were purchased from animal center of Southern Medical University. Focal cerebral ischemia was produced by intraluminal MCAO following the method of Longa et al. After fasting overnight, the animal was lightly anesthetized with Chloral hydrate, The left common carotid artery and the left external carotid artery were exposed, and the occipital artery and superior thyroid artery were electrocoagulated. The left pterygopalatine artery was ligated, the left common carotid artery was clamped, and a 4-0 nylon suture was inserted retrogradely via arteriectomy of the external carotid artery into the internal carotid artery. The tip of the suture was rounded near a flame before insertion. The suture was slowly advanced until resistance was felt. The external carotid artery was ligated, and the common carotid artery clamp was released. The skin incision was sutured.The animal was allowed to recover and was kept warm with a heating lamp. The rat was sedated with halothane and killed by decapitation at 24 hours.Neurological status before the animal was killed was measured as described by Longa et al. Some animals in each treatment group died during the night; these animals were replaced in the study and were not included in the calculation of infarct area.2) A preliminary study of the therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemiaPermanent embolism in 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain slices staining by TTC, comparing the area of cerebral infarction embolization 1 hour delivery model group and model group,the study showed BDNF-PTD can protect ischemic neurons in the brain.3) The Mechanism Of Reversal the neuron death on brain ischemia by BDNF-PTDBDNF can enhance the capacity of neurons to resist ischemia and promote repair of damaged neurons, regeneration, regulate neural structure, reconstruction, the promotion of cognition functional recovery after brain injury. The mechanism of its role may be related to the two main signaling pathways, these two signaling pathways has been proven to promote the growth and survival of neurons. Phosphatidylinositol-3-kinase (PI3K) pathways and cytokinins (mitogen)-activated protein kinase (MAPK) signaling pathways, which through activation transcription proteins (such as the cAMP response element binding protein) affect gene expression to promote nerve growth and survival. The mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathway with the BDNF-TrkB receptor signaling. BDNF-PTD has a reverse function of neuronal death, whether it is through these pathways play a role, which will verify in this paper.Permanent embolism after 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain were dissected, complemented with lysis buffer, homogenized, and centrifuged, and supernatants were used for Western blot (WB), verify whether the BDNF-PTD can activate ERK extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K).RESULTS1) The establishment of SD rat model of cerebral ischemia Cerebral ischemia model is made by middle cerebral artery occlusion (MCAO), the activities in the neurological function have different performance in the durg group and the blank model group, negative control group when 24 hours after vascular occlusion. range of deficit score 0 to 1 in the treatment group. The model group and negative control group, decreased resistance to thrust and with the contralateral forelimb flexion, and turning in circles rear-end movements(range of deficit score 2 to 3).2) The preliminary study of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemiaThe hemiplegic symptoms are significantly improved in thrombosis 1 hour group by middle cerebral artery occlusion after administration BDNF-PTD, rather than non-treatment group. TTC stained brain slices and found rat brain necrotic area of 1 hour thrombosi group has significantly reduced compared with the control group. Infarct size Values given are mean±SD, treatment group compare to no treatment group with negative control group, the results statistically significant difference (F= 26.791, P= 0.000<0.01). The necrotic area of treatment model group are significantly reduced(75%～80%) compared with nontreatment model group.Results show that BDNF-PTD can through the blood-brain barrier and can perform its biological effects compared with the control group, this result found that BDNF-PTD can be well protected neurons survive after cerebral ischemia. 3) The mechanism of therapeutic effect brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemiaStudies the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. Focal cerebral ischemia model in rats produced by middle cerebral artery occlusion (MACO). intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD after Permanent embolism in 1 hours, after 24 hours the animal decapitated and the brain protein analysis by Western blot, results show BDNF-PTD activated ERK through phosphorylation, pERKprotein levels were significantly increased (P<0.01) compared with normal group; and nontreatment model group, pERK protein levels also were significantly elevated (P<0.05).In addition, results also shows, BDNF-PTD could were significantly upregulate PI3K (P=0.000<0.01) compared with normal group;PI3K levels also also were significantly elevated (P=0.000<0.01)compared with nontreatment model group.but the PI3K levels were not statistically significant (P>0.05) in nontreatment model group between normal group.CONCLUSION1) The embolism hemiplegia symptoms are significantly improved in middle cerebral artery occlusion model after treat by BDNF-PTD. Study found that brain necrotic area were significantly reduced in treatment group compared with the control group. As compared with the model group, BDNF-PTD can pass through the blood-brain barrier and perform its biological effects which demonstrate that BDNF-PTD can be well protected neurons in cerebral ischemia.2) To study the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. produced middle cerebral artery occlusion (MACO) model through Suture method in rats. after 24 hours the rats decapitated and brain protein analysis by Western blot, the results confirmed that the BDNF-PTD can activated ERK extracellular signal-regulated kinase and upregulate phosphatidylinositol 3-kinase (PI-3K).