| BackgroundAlzheimer's disease is one of the most common types of dementia,accounting for 50%to 70%of the total dementia population.Its concealed onset seriously affects the daily social and occupational functions of patients.Nowadays,the clinical diagnosis of AD is mostly based on the symptoms of cognitive impairment,but for the elderly worldwide,it is necessary to pay attention to other factors related to the disease process,and further explore early diagnosis and intervention methods.In recent years,more and more evidences have shown that olfactory dysfunction of AD patients is highly correlated with the progression of their disease,and olfactory pathways were found with pathological changes in early stage.Therefore,olfactory dysfunction may be a potential target for early diagnosis and treatment of AD.Olfaction is the only sensation in which the external environment directly connects the brain through the central nervous system.It is not only closely related to animal survival instincts such as foraging,reproduction and vigilance,but also regulates important senior functions such as emotion,learning and memory ability,visceral activities and so on.There are both innate and acquired associations between olfaction and memory.In anatomy,olfactory systems are closely related to brain areas of learning and memory.Functionally,olfactory pathways can affect synaptic remodeling through odor stimulation,thereby regulating learning and memory ability.Olfactory impairment can also accelerate the development of neurological dysfunction,leading to cognitive decline.In addition,Olfactory dysfunction is associated with a variety of neurodegenerative diseases.As a route of administration,olfactory pathway has unique advantages to some central nervous system drugs.Therefore,the relationship between olfaction and memory has gradually become the focus of researchers in recent years.Aromatherapy is a natural therapy that uses aromatic drugs or plant preparations to keep mind and spirit balanced.In recent years,researchers have found that aromatherapy has certain advantages in the treatment of AD.It can improve learning and memory ability,lesson emotional abnormalities and aggressive behavior of AD patients without obvious side effects.The mechanism is to stimulate the olfactory system with aromatic substances.Moxibustion therapy has been used in the field of health preservation since long time ago.Its characteristics of tonifying Yang Qi,warming and dredging channels and collaterals enable it to perform well in the treatment of geriatric diseases.As a combustion product,moxa smoke is the substantial basis of the pharmacological effect of moxibustion therapy,and has some similarities with aromatherapy.Our previous studies found that the independent intervention of moxa smoke can improve the learning and memory ability of mice,regulating the inflammatory response and oxidative stress in the brain.The approach of moxa smoke may be respiratory tract,olfactory pathway or skin,but researches on the mechanism of moxa smoke are lack at home and abroad,especially on the pathway of moxa smoke.With senescence accelerated mouse P8(SAMP8)as model,3-methylindole(3-MI)intraperitoneal injection as modeling method,neurobehavioral experiments and morphological observations were carried out to investigate the effects of moxibustion on spatial learning and memory of mice through olfactory pathway,so as to lay a foundation for further exploring the mechanism of moxa combustion products.Objective To observe the effects of moxa smoke inhaling on learning and memory ability,morphology of hippocampus and olfactory bulb,and the trend of neurotransmitters variation in olfactory bulb and hippocampus of SAMP8 at the basis of olfactory pathway,in order to explore the pathways and possible mechanism of moxa smokeMethod 48 6-month healthy male SAMP8 of SPF grade were randomly divided into moxa smoke group,dysosmia with moxa smoke group(dysosmia smoke group),dysosmia group and model group.12 SAMR1 were used as normal control group.Modeling method:3-MI was dissolved in corn oil to a concentration of 30 mg/ml,then injected intraperitoneally to mice in dysosmia smoke group and dysosmia group at a dose of 300 mg/kg.Buried food pellets test was carried out to test model at 24h after administration.Intervention:Moxa smoke group and dysosmia smoke group:Fine moxa sticks were ignited and fixed in the middle of the partition of the smoke inhaling box.After the concentration of moxa smoke reached predefined standard(5-15mg/m),mice were put into the box,30 minutes/day,6 weeks.The dysosmia group,model group and normal control group inhaled normal air without special treatment.After 6 weeks of intervention,open-field test and Water Maze experiment were taken,and then the mice were executed.The pathology change of hippocampus and olfactory bulb were observed by HE staining.Elisa method was used to detect the contents of Glu,GABA,DA and 5-HT in hippocampus,and Glu and GABA in olfactory bulb.Result1.Modeling:After modeling and six-week intervention,the average searching time of BFPT experiment in the dysosmia group and dysosmia smoke group were beyond 300 seconds.2.Open field experiment:Compared with the normal group,horizontal and vertical motion scores in the other four groups were significantly higher(p<0.0.1),and the residence time in the central area of model group was shorter(p<0.05).Compared with the model group,the vertical motion of dysosmia group was significantly increased(p<0.05),and the residence time of central area was shorter(p<0.05).Compared with dysosmia group,the residence time in the central area of moxa smoke group increased significantly(p<0.05),and that of dysosmia smoke group had a rising trend(p>0.05).There was no significant difference in fecal quantity among groups(p>0.05).3.Morris water maze experiment:In the orientated navigation stage,the escape latency and total distance of dysosmia smoke group was lower than model group in day3 and day4.In day3,total distance of the dysosmia group was higher than dysosmia smoke group,and the latency was higher than normal group and dysosmia smoke group in day4.In the stage of space exploration,the residence time of target quadrant of moxa smoke group was longer than model and dysosmia group,and its swimming distance ratio was higher than model group.4.Morris water maze experiment:The escape latency time of model group from Dayl to Day4 was always higher than that in normal group(p<0.05).The latency of day3 and day4 in moxa smole group was significantly lower than that in model group(p<0.05),while the latency of moxa smoke group were significantly lower than that of model group,but there was no statistical significance(p>0.05).Compared with the dysosmia smoke group,the latency of dysosmia group was significantly higher in Day4(p<0.05).Total swimming distance of dysosmia smoke group in Day3 was significantly lower than that of dysosmia group(p<0.05),and in Day3 and Day4 it was lower than that of model group(p<0.05).In the stage of space exploration,the residence time of target quadrant in moxa smoke group was longer than that in model group and dysosmia group,and its swimming distance ratio was higher than that in model group(p<0.05).5.HE staining:Olfactory bulb:The olfactory bulb of mice in each group had clear structure,regular arrangement and no obvious abnormality of neurons were observed.Hippocampus:The hippocampal neurons showed regular shape,dense neurons with plenty cytoplasm and deep-dyed big nucleolus were observed.The number of hippocampus neurons of dysosmia smoke group decreased significantly,with triangular or polygonal shape,irregular arrangement and loose nuclear chromatin.The cell margins were blurred.And some nuclei deformed,shrinked or disappeared.The structural disorder and degeneration of hippocampal neurons in the dysosmia group were more serious than other group.Compared with the dysosmia smoke group,dysosmia group and model group,the hippocampal neurons of the moxa smoke group were relatively dense,with deeper stained nucleus.The whole neural arrangement was more orderly,and the cell edge structure was relatively complete and clear.6.Neurortransmitters in hippocampus and olfactory bulb:Glu in hippocampus and olfactory bulb of model group was higher than normal group(p>0.05).Glu in hippocampus and olfactory bulb of moxa smoke group was lower than that of model group(p<0.05,p<0.01),and Glu of moxa smoke group was lower than dysosmia smoke group and dysosmia group in olfactory bulb(p<0.05);GABA in hippocampus and olfactory bulb of normal group was significantly higher than model group,and there was no significant difference among the other groups.Compared with model group,DA in hippocampus of moxa smoke group,dysosmia smoke group and dysosmia group increased significantly(p<0.01).5-HT in hippocampus of moxa smoke group was higher than dysosmia smoke group and model group(p<0.05).Conclusion(1)Certain concentration of moxa smoke can improve the spatial memory of SAMP8 through olfactory pathway,but olfactory pathway is not the only way of moxa smoke effect.(2)Moxa smoke can decrease the abnormal accumulation of Glu in olfactory bulb and hippocampus via olfactory pathway,increase DA and 5-HT in hippocampus,and improve the disorder of neurotransmitters in brain,thus protecting neurons and delaying the pathological progress of AD.(3)The changes of neurotransmitters in hippocampus and olfactory bulb were consistent after moxa smoking,suggesting the mechanism of moxa smoke may be mediated by the indirect projection between olfactory bulb and hippocampus with the changes of excitatory neurotransmitters. |
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