Protective Effect And Mechanism Of Volatile Oil From Acorus Gramineus On Neuron Injury In AD Model Mice

Alzheimer’s disease（AD）is a kind of onset conceals,primary degenerative disease of the nervous system,commonly known as alzheimer’s,mainly manifested as progressive language,memory impairment;personality change;cognitive dysfunction and other neuropsychiatric symptoms,which seriously affects the patient’s career,normal social interaction and life function.Its characteristic pathological changes are cells of the beta amyloid protein（Aβ）are deposited in the extracellular senile plaques and neurofibrillary tangles（NFT）,etc.The"A beta hypothesis",which is due to the abnormal metabolism of the body,Aβcan not be removed in time to the specific region of the brain,causing the death of neurons,leading to AD.Acorus gramineus is a perennial herb Araceae,it is also known as the dried rhizome of Acorus gramineus,spicy and warm.It has the effect of opening the orifices and phlegm,transforming the wet stomach and awakening the mind and the wisdom.Widely used in clinical treatment of epilepsy,Alzheimer’s disease,stroke,tinnitus forgetful aphasia and difficult miscellaneous diseases.The extraction site of volatile oil from Acorus gramineus has significant effect on anti-dementia and improving the learning and memory of animals.In recent years,more and more studies have been done in the nervous system of Acorus gramineus,but little research has been done on the volatile oil from Acorus gramineus,and there is less research on the therapeutic effect of volatile oil from Acorus gramineus on AD.This study used the volatile oil from Acorus gramineus for the experimental study of AD model mice,to observe the protective effect of the volatile oil from Acorus gramineus on AD model mice neuron injury and the possible mechanism,to provide the experimental basis for the volatile oil from Acorus gramineus improvement and treatment of AD.Objective:This study investigated the protective effect of the volatile oil from Acorus gramineus on the neuron damage in AD model mice,and to study the effect of volatile oil from Acorus gramineus on the improvement of AD and the related mechanism.Method:Dry rhizome of Acorus gramineus was be used,and smashed by grinder,through 2 sieves（20 mesh）.200 g Acorus calamus crude powder was added in each bottom flask with 10 times amount of water,soaked for 1h,and added several zeolites,steam distillation to extracted volatile oil from Acorus gramineus,and stopped heating when the volatile oil no longer increased.The KM mice were divided into four groups:normal group,control group,model group（Aβgroup）and treatment group.The treatment group was further divided into treatment group I:Aβand per 10g body weight 2.5μL volatile oil from Acorus gramineus,and treatment group II:Aβand per 10g body weight5μL volatile oil from Acorus gramineus.Model group and treatment group were injected with oligomeric Aβ_1-42-42 by cerebral stereotactic apparatus in the bilateral hippocampus of KM mice to established the AD models.The control group was injected the same amount of saline with the same method,and the normal group did not deal with it.On the seven day after modeling,the mice with cognitive dysfunction were selected by the novel location recognition（NLR）task.After that,the treatment group began to intragastric administration for per 10g body weight2.5 or 5μL volatile oil from Acorus gramineus,the control group and the Aβgroup were given the same amount of saline,and the medicine was administered for three weeks.The novel location recognition（NLR）task was done again,and the cognitive function of each group after the volatile oil from Acorus gramineus was analyzed.HE staining was used to observe the changes of cell morphology in the hippocampus of mice in different groups.Immunohistochemistry was used to detect the deposition of Aβ_1-42-42 in the hippocampus and the expression of DCX Nestin、BDNF、TrkB and NT3 in the hippocampus of different groups.Western blot was used to detect the expression of Nestin、DCX、BDNF、NT3 and TrkBResult:1、The results of the novel location recognition（NLR）task showed that compared with the normal mice,the cognitive function of the control group did not change significantly（P>0.05）,while the cognitive index of the model group decreased significantly（P<0.01）,indicating that its cognitive function was significantly reduced.Compared with the model group,the cognitive index of the mice in the treatment group was obviously increased（P<0.05）,that is,the cognitive function of the mice in the treatment group was improved.2、The results of HE staining showed that compared with the normal group,there was no serious damage to the cells in the hippocampus of the control group,but the cells in the vicinity of the needle channel were arranged in disorder.Compared with normal group and control group,the cell of hippocampus in model group was seriously damaged,and the change of hippocampus dentate gyrus cells was mainly manifested by nuclear deep staining,nuclear shrinkage,loosening of cell arrangement,and cell body detached from surrounding tissues,compared with the model group,the treatment group improved.3、The immunohistochemical results of Aβshowed that Aβdeposition was almost impossible in normal group and control group,and there was more Aβdeposition in the hippocampus of model group.Compared with the model group,the content of Aβ_1-42-42 in the hippocampus of the treatment group was significantly lower than that in the treatment group,and the statistical results have significant differences.4、The immunohistochemical and Western blot results of DCX、Nestin showed that there was no significant difference in the content of DCX、Nestin in the brain of the control group compared with the normal group（P>0.05）.However,in the model group,the expression of DCX、Nestin in the brain of the mice decreased significantly,and the statistical results have significant differences（P<0.05）.Compared with the model group,the expression of DCX was significantly increased in the treatment group（P<0.05）.5、The immunohistochemical and Western blot results of BDNF and TrkB showed that there was no significant difference in the contents of BDNF and TrkB in the brain of the control group compared with the normal group（P>0.05）.In the model group,the expression of BDNF and TrkB in the brain of the mice decreased significantly,and the results have significant differences（P<0.05）.Compared with the model group,the expression of BDNF and TrkB in the brain of the mice was significantly increased in the treatment group,and the results have significant differences（P<0.05）.6、The immunohistochemical and Western blot results of NT3 showed that there was no significant difference in the contents of NT3 in the brain of the control group compared with the normal group（P>0.05）.In the model group,the expression of NT3 in the brain of the mice decreased significantly,and the statistical results have significant differences（P<0.001）.Compared with the model group,the expression ofNT3 in the brain of the mice was significantly increased in the treatment group,and the statistical results have significant differences（P<0.01）.Conclusion:1、The microinjection of Aβ_1-42-42 into the hippocampus could induce a series of AD related pathological changes and cognitive dysfunction in mice brain.2、The volatile oil from Acorus gramineus could improve the cognitive function of AD model mice.3、After treatment of volatile oil from Acorus calamus,the number of DCX and Nestin positive cells increased significantly,suggesting that the volatile oil of Acorus gramineus may induce the regeneration of hippocampal neurons in mice;and it may promote and protect the growth of hippocampal neurons by up regulation of BDNF,TrkB and NT3 expression.