| BeckgroudCerebral ischemic is the most life-threatening neurological disease among the elderly, and is the third-leading cause of death in a lot of the countries in the world. The human costs of stroke are very large and growing; and survivors are often faced with loss of ability to function independently. Many studies have done at home and abroad for the disease pathogenesis ,and pharmaceutical research of this disease has made a lot of progress. There are some new drugs for the treatment of cerebral ischemia, such as the thrombolytic compound tissue plasminogen activator (t-PA) and so on. But, these neuroprotective drugs in clinical application performance is often unsatisfactory. In addition, many of them have lot of adverse reactions or therapeutic window is unsuitable for many cerebral ischemia. Looking for the new targets and drugs for the treatment of stoke remains a priority. It is most likely that the original Sumerian word"kunibu"developed into the forms"kan(n)ab(is)". the first written note about the medical use of cannabis was also discovered in China, which dates back to 2727 B.C. Seeds of cannabis were found in 8,000 years old Chinese food remains. Cannabis seems to be responsible for the vast majority of effects such as motor disturbances and catalepsy, corneal areflexia (in rabbits), scratching, euphoria and dysphoria, anxiety, drowsiness, altered time and audiovisual perceptions, panic attacks and impaired memory Both its principal active ingredient,△9-tetrahydrocannabinol(△9-THC), and synthetic analogs thereof(cannabinoids)have been proposed as herapy for a variety of medical conditions,including glaucoma , cancer chemotherapy-induced nausea and vomiting,acquired immunodeficiency syndrome,inflammatory disorders,and epilepsy. But,they share effects with narcotics and their possible role in infertility destroy the enthusiastic for further study of cannabis drugs.Both cannabinoid receptor are the most abundant G protein-coupled receptor (GPCR) which mainly existing in the mammalian brain and seven transmembrane-domain receptors of the rhodopsin type. The distribution of CB1 receptors in neurons localized to corticolimbic areas, basal ganglia, cerebellum, and brain-stem. The CB1 receptor is the predominant cannabinoid-sensing receptor subtype expressed on neural cells in the central nervous system (CNS). In contrast ,the CB2 receptor are commonly associated with the regulation of immune function. The CB1 receptor in the brain mainly found in the globus pallidus, striatum, substantia nigra, hippocampus, cerebellum and cerebral cortex were higher in the density of the CB1 receptor. In addition, in testis, vas deferens, uterus, lung, small intestine, vascular smooth muscle cells and endothelial cells were found in the presence of the CB1 receptors also. CB2 receptor is mainly distributed in the spleen, tonsils, lymph nodes and other immune organs and in macrophages / monocytes, B lymphocytes, NK cells (natural killer), eosinophils and white blood cells with high expression. The first endocannabinoid to be found was anandamide that was isolated from pig brain by Devane and coworkers (1992). Two types of arachidonic acid-containing lipid molecules, anandamide (N-arachidonoylethanolamine, AEA) and 2-AG (sn 2-arachidonoylglycerol), have been identified as the endogenous ligands (endocannabinoids) for the cannabinoid receptors. Anandamide and 2-AG binds to the cannabinoid receptors with high affinity and exhibits a variety of cannabimimetic activities as△9-THC. The two endocannabinoid to be found have the same arachidonic acid derivatives structure. Endocannabinoids, and the cannabis receptors constitute the ECS (The endocannabinoid signaling system). the onset of the boom was there when the structure of THC and the cloning of the first cannabinoid receptor from human and rat being reported. The discovery of ECS declared a new enkephalin era already on the horison.Anandamide was found widely in many mammalian tissue, such as brain, heart, spleen and uterus and other organs and with complex physiological effects. In some metabolic disorders or diseases, the ECS effect has been a hot issues. There are conflicting reports in the literature describing the influence of cannabinoid receptor activation on a lot of diseases. Whether cannabinoid receptor activation by modulating both neurotransmission (CB1 ) and neuroinflammatory (CB2 ) responses have neuroprotective effect during I/R is a new trends in ECS research field.Objectives and Methods(1) To estabilish a sensitive and selective analytic method determining the concentration of anadamide in the mammal and human beings body fluid using reversed-phase high performance liquid chromatography(HPLC) fluorescence detection with pre-column derivatization.(2) To determine the distribution of endogenous ligand anandamide(N-arachidonylethanolamine,AEA) in different organ tissues and body fluid from mammals and human beings by the precolumn derivatization of anandamide with DBD-COCl , separation using reversed-phase high-performance liquid chromatography and detected by fluorescence detection.(3) Because the role of endocannabinoid signaling in the response of the brain to injury is tantalizing but not clear. In this study,transient middle cerebral artery occlusion(MCAo)was used to produce ischemia/reperfusion injury and the concentration of ananadamide was detected by the method we established. The effect of concentration changes on AEA and the role of the endocannabinoids in the outcome of a transient,focal chemic/reperfusion will be discussed.(4) The goal of this study was to regulate the balance between CB1 and CB2 activation following cerebral ischemia influences outcome by drug combination.Results and Conclusions(1) The analyte was separated using HPLC after derivatization with 4-(N,N-dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methyl-amino)-2,1,3-benzoxadiazole(DBD-COCl). Established pre-column derivatization HPLC analysis can be successfully detected for anandemide in different tissues and body fluids from mammal and human beings. The endocannabinoid anandamide, not only in the central nervous system but also in peripheral tissues, cannabis endogenous ligand anandamide concentration were significantly different in different organs and body fluids,which may regulate multiple functions of the endocannabinoid sysytem.(2) During a brief period of focal ischemia brain endocannabinoid content increase significantly(p<0.01).The role of the endocannabinoids in the outcome of a transient,focal ischemic/reperfusion is neuroprotective effect. There was a redistribution of AEA from the ipsilateral to the contralateral cortex during this period resulting in an overall elevation of AEA in whole brain.(3) The combined CB1 antagonist and CB2 agonist had significantly improved motor function compared to the control group(p<0.05).In parallel with the changes in neurological function, administration of the Cantagonist plus CB2 agonist 1 hour before MCAO dramatically reduced infarct volume(p<0.05) and rCBF increased during the occlusion period. The combination of a CB1 agonist and a CB2 antagonist provided the neroprotection effect and indicated a synergistic effect derived from combining these agents. CB2 cannabinoid agonists protected against I/R injury by decreasing the endothelial cell activation/inflammatory response, the expression of adhesion molecules, the modulation of inflammatory cytokines/chemokines levels, recruitment, adhesion and activation of inflammatory cells, and interrelated oxidative/nitrosative stress .CB2 receptor activation also attenuates TNF-α-induced NF-kB and RhoA activation, up-regulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein in human coronary endothelial cells, enhanced transendothelial migration of monocytes, and augmented monocyte- endothelial adhesion.Therefore, changing the balance of stimulation of these receptors by endogenous cannabinoids may provide an important therapeutic strategy during stroke. |
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