| Objective The treatment of cerebral ischemia disease in clinical is long-term oral anticoagulation drugs or expansion of blood vessels. The mechanism of ischemic stroke in clinical mainly includes Oxidative damage, the release of immune inflammatory mediators,or the lack and release of neurotrophic factors, etc.In previous studies we found that the aromatic small molecules can correspond the neuron receptors through the olfactory pathway,to adjust the excitement or inhibition of nerve cells.It may be a new way of intervention or treatment for ischemic stroke.The human sense of olfactory is closely related to brain function.The sense of olfactory affect the life instinct, the ability of learning and memory, emotion regulation, etc.Olfactory is closely related to human survival quality, many serious diseases are associated with olfactory dysfunction early.Olfactory adjust to the cerebral cortex, hippocampus, amygdala, hypothalamus, striatum, piriform cortex and entorhinal cortex of brain regions. Is there a link between the lack of cerebral stroke and the olfactory pathway ?The method of inhaling is more convenient and safe, high specificity and high sensitivitythan than oral dosing and drug injection delivery.What is the mechanism of aromatic molecules through the olfactory pathway?In this experiment we explore the protection after cerebral ischemia injury and its mechanism on Eugenol Inhaled through olfactory pathways in MCAO model rats.Part One Establish the MCAO Rats Model And Score the Nerve FunctionMethodsEstablish the MCAO rats model after ischemia 2h and reperfusion 24 h.Reference to Longa evaluation standard to score the nerve function, the score of 1 to 3 is divided into the experiment rats,random grouping: model group, eugenol group, Sham group and normal group.ResultsModel and eugenol group nerve function score is 1 to 3 points.The rats significantly in circles, dumping,and forelimbs can’t stretch.The nerve function score of Sham group and normal group is zero, no any abnormal behavior.Part Two The Intervention for Eugenol Inhaled in MCAO Model Rats and the Observation of Neuroethology BehaviorMethodsEugenol group was given eugenol inhaled after 3w, the rest of the group do not make deal with normal feeding, to score nerve function by each group again, Executed, the brain takes TTC staining, calculating the cerebral infarction volume percentage; Testing the ability of learning and memory by the Morris water maze experiment.ResultsNerve function score: eugenol group compared with model group, with significant difference(P<0.01);After ischemia/reperfusion rats are not degree of ischemic infarction, the infarcts area/brain slice area of eugenol group significantly decreased compared with model group, with significant difference(P < 0.01);The Morris water maze experiment:in navigation experiments, the escape latent period of model group was obviously longer than normal group and sham group(P<0.05);And the escape latency period of eugenol group was shorter than the model group, significant differences(P<0.05);Part Three The Observation of Nerve Morphology and the Influence on 5-HT and BDNF of Eugenol InhalationMethodsThrough neuroethology test can show eugenol inhalation can improve learning and memory after ischemia rats.To explore the mechanism of eugenol,We execute the rats and put the brain section into HE staining to observe the cell morphology.Observing the changes of 5-HT and BDNF expression in brain area by immunohistochemical method.Finally we analysis the positive reactant average optical density of BDNF and 5- HT in hippocampal CA3 area.ResultsHE staining showed the sham group and normal group cells were quantity, order, the form was circular or elliptic, structure was clear;The number of cells in model group decreased, shape was small and irregular, structure was not clear. The number of cells in model group decreased,the shape was small and irregular,the structure was not clear; The number of cells in eugenol group increased, the structure was clear.Immunohistochemical results showed 5- HT and BDNF reactants was tan, the immune responses of 5- HT and BDNF in sham group and normal group was strong positive, eugenol group was medium, model group was weak, and number of cells was less.The expression of 5-HT and BDNF protein of the average optical density in hippocampal CA3 area :eugenol group, normal group and sham group compared with model group of 5- HT average optical density(P < 0.01),BDNF average optical density(P < 0.05),, the difference was statistically significant.Part Four Explore the Mechanism of Eugenol Through Reverse Virtual ScreeningMethodsUsing molecular docking software Surflex, eugenol, for a active small molecule probe, and docking in sc PDB database with 8077 protein crystal virtual, find out the most likely protein receptors to combine, analysis the mechanism of the drugs, to provide theoretical guidance for the next experiment.Reverse the virtual screening results not only can be systemic summarized previous research,but also to forecast the potential effects of eugenol targets.ResultsThrough the Surflex_scoring, screening t h e t o p 2 0 p r o t e i n c r y s t a l s as eugenol medicinal targets.Mainly include carboxyethyl arginine beta lactamase, thrombin, methine courageous alkanes urine bile pigment in the original four hydrogen hydrogenase, salicylic acid synthetase oxidoreductase tuberculosis, etc. the top 20 score protein crystals are shown in table 4.The effect of anti-inflammatory and antioxidant is the same as we know.The combination of thrombin and eugenol mode as shown in figure 11 D, because thrombin can anticoagulate and inhibit platelet aggregation, it could be a potential mechanism of eugenol for cerebral ischemia injury.Conclusions1. The olfactory pathway is closely related to cerebral ischemia. 2. Eugenol Inhalation via olfactory pathway can improve ischemia injury in MCAO model rats and its mechanism may be related to the expression of BDNF and 5- HT in brain hippocampus. 3. Through the computer reverse virtual screening of eugenol targets, part of the screen targets are the same as pharmacological action reported, of which the binding sites of and thrombin may be related to cerebral ischemia injury. |
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