| BackgroundAlzheimer's Disease(AD)is a chronic progressive disease with latent morbidity.From now on,it is the most common degenerative disease of the central nervous system.Its main clinical manifestations are memory loss,cognitive dysfunction and personality changes.At present,the cause of AD is unknown,and the pathogenesis is unclear.On the etiology of AD,there are ? amyloid protein(A?)cascade hypothesis,tau protein abnormal modification theory,neuronal apoptosis theory and gene mutation theory.The A? deposition in the brain and the toxic effects of senile plaques on neurons,are recognized by almost all hypotheses.A? is a cleavage product formed by cleavage of amyloid precursor protein(APP)by ?-secretase and y-secretase.An imbalance between clearance and production of A? leads to its accumulation in the brain,and results in a decrease in the ability to clear A? is the main cause of its deposition.The clearance of A? in the brain is mainly accomplished through the blood-brain barrier pathway,which is transported from the brain into the peripheral blood circulation through the basal and basal surface receptor proteins of the brain capillary endothelial cells(BMECs).Complete removal;several batches of experiments in the group were mainly to observe the relationship between changes in A? levels and the basal surface receptor protein Low density lipoprotein receptor-related protein 1(LRP1).However,this experiment now focuses on the P-glycoprotein(P-gp),a hot spot protein currently under investigation,to observe its positive expression in the hippocampus and its relationship with changes in A? levels.Brain A? is mainly cleared through the brain blood-brain barrier(BBB)pathway.This process requires the participation of BMECs.P-protein is highly expressed on its basal layer.Interstitial fluid(ISF)is the intermediate of A? into different metabolic pathways.Sites,observations of ISF A? can provide an overall assessment of brain soluble A? levels.With the gradual intensification of the ageing trend,common diseases in the elderly have become more and more concerned by the society.Epidemiological studies have shown that AD is the second most important problem faced by geriatric medicine,which is second only to cardiovascular diseases,cancers,and strokes that threaten the life and health of the elderly.The incidence of AD become more and more popular and younger.However,there are currently no specific drugs that can treat AD.Currently,the treatment methods used are mainly to alleviate various symptoms,including mental symptoms,which occur during the course of the disease,and to delay the further development of dementia.Once the severity of cognitive impairment reaches the standard of dementia,it will be very difficult to reverse,and the latest treatment concept will be to intervene in the preclinical period of AD,thus delaying the occurrence of symptoms.Therefore,during the clinical treatment of AD disease,we adopt more comprehensive treatment methods,such as adding traditional Chinese medicine,acupuncture therapy,etc.in order to explore more ways to treat AD disease.Acupuncture and moxibustion treatment is very popular in China,and the time and money spent on acupuncture treatment can be widely accepted by the people.Chinese acupuncture and moxibustion treatment of encephalopathy has a history of thousands of years,and the advantages of acupuncture in the treatment of AD with multiple layers,multiple pathways,and multiple target sites all show that acupuncture treatment is positive for the efficacy of Alzheimer's disease.The mechanism of action provides a broad idea.ObjectiveIn this study,low-month-old double-transgenic APPswe/PS1dE9 double transgenic male mice were selected and treated with short-term electroacupuncture for their "Baihui and Yongquan points" based on the traditional Chinese medicine treatment mechanism of "Feizhen Guben and Tongluo Turbidity." Using brain microdialysis detection technology to take the brain interstitial fluid in mice to observe whether the level of A?1-42 in brain interstitial fluid changes under the action of electroacupuncture.The hippocampus was sampled and observed by morphological and semi-quantitative methods to determine whether the level of P-glycoprotein in the hippocampus was altered by electroacupuncture stimulation and whether there was a correlation between changes in A?1-42 and P-glycoprotein.To explore the effect of early electroacupuncture intervention on the levels of interstitial fluid A? and hippocampal P glycoprotein in APP/PS1 transgenic mice,and the correlation between them.MethodIn this experiment,18 4-month-old male APP/PS1 double transgenic mice were selected as animal models of AD and were randomly divided into two groups based on random number tables---electroacupuncture treatment group(A)and model group(M).9 in each group.Nine male 4-month-old C57BL/6 mice served as normal controls(C).Experimental animals were purchased from the Institute of Animal Research in Nanjing University.The experiment was approved by the Experimental Animal Ethics Committee of the Beijing University of Traditional Chinese Medicine.The mice in the acupuncture group were fixed on the operating table with a self-produced rat bag.According to the acupuncture point map and comparative anatomy methods of the rat,the "Baihui" was taken in the center of the parietal bone and was taken at the anterior and middle 1/3 of the two foot soles."Yongquan." The disposable medical acupuncture needle(Beijing Zhongyan Taihe Pharmaceutical Co.,Ltd.,size:0.25 mm×13mm)was used.The points and needles were routinely sterilized.Acupuncture points were inserted at an angle of 15° to a depth of 2-3 mm under the skin.The "Yongquan point" was slightly twisted and then connected to the Korean electric needle instrument.The density was 1/50Hz and the intensity was 0.1 mA.The needle shanked and the animals remained quiet and did not struggle.Each time the acupuncture lasts for 15 minutes,every other day,7 times.Rats in the model group and the normal control group were tied with a mouse bag for 15 minutes in the same manner,every other day,totaly 7 times.After the intervention of acupuncture,brain interstitial fluid was taken by brain microdialysis technique.The level of A?1-42 in hippocampal interstitial fluid of APP/PS1 mice at 4 months of age was detected by Elisa method,and the effect of electroacupuncture was observed.After the end of microdialysis surgery,the mice were anesthetized,and 3 mice were randomly selected from each group to be anesthetized with cardiac perfusion.The hippocampus was selected for resin embedding and immunohistochemistry was used to observe the positive expression of hippocampal P-glycoprotein.The relationship between P-glycoprotein and interstitial fluid A?1-42 levels was observed,and electro-acupuncture was used for its effect,In addition,the other 6 mice in each group were decapitated and its hippocampus was reserved.Western blot was used to observe the hippocampal P-glycoprotein level and the intervention of electroacupuncture.ResultsImmunohistochemical detection of P-glycoproteinInununohistochemistry was used to treat paraffin sections of hippocampus in mice of different groups and the expression of P-glycoprotein was observed under a 400-fold magnification using a microscope.It was found that in the same region of the hippocampus,positive expression of P-glycoprotein was observed in all three groups.Both are expressed on the microvascular wall.The normal control group had the largest positive expression area and darker color;the positive expression area of the model group decreased significantly and the color also became lighter;compared with the model group,the positive expression area of the electroacupuncture treatment group get increased.Semi-quantitative analysis of IOD values for positive expression of P-glycoprotein was tested by IPP software.Compared with the normal control group,the P-glycoprotein IOD value of the hippocampus in the model group was lower;compared with the model group,the hippocampal P-glycoprotein in the electroacupuncture group was higher.ELISA detection of interstitial fluid A? levelsCerebral interstitial fluid in each group of mice was sampled by brain microdialysis and the levels of A?1-42 were detected by ELISA.The level of A?1-42 was significantly increased in the model group compared with the normal control group(P<0.01).and the level of A?1-42 was decreased in the electroacupuncture group compared with the model group,the difference was statistically significant(P<0.01).This shows that the short-term treatment of electroacupuncture has the effect of reducing the interstitial fluid A?1-42 in the brain.Western blot detection of hippocampal P-glycoproteinSamples of fresh hippocampal tissue from mice were prepared by adding RIPA lysate,and the level of P-gp in hippocampus was detected by Western blot.The level of P-gp in the model group was significantly lower than that in the normal control group(P<0.05).Compared with the model(M)group,the level of P-gp was increased in the electroacupuncture group and the difference was statistically significant(P<0.05).This shows that electro-acupuncture treatment can promote the expression of hippocampal P-gp.Correlation AnalysisThe correlation analysis between mouse brain interstitial fluid A(31-42 and hippocampal P-glycoprotein level indicators showed that there was a negative correlation between mouse brain interstitial fluid A?1-42 levels and hippocampal P-glycoprotein levels(r=-0.69,P ? 0.01),and showed a strong correlation,and the results' difference was statistically significant.ConclusionFour-month-old APP/PS1 double transgenic mice successfully mimicked the elevation of A? in the pathological changes of AD and decreased levels of the related transporter P-glycoprotein.After electroacupuncture treatment,the level of interstitial A? in brain tissue is decreased,which may be related to the increase of P-glycoprotein expression in the hippocampus.This may be one of the ways in which electroacupuncture can treat AD. |
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