Study On The Effect And Mechanism Of β-asarone On APP/PS1 Transgenic Mice Based On BACE1 Target And PINK1/Parkin Mediated Mitophagy

Alzheimer’s disease(AD)is the most common pathological type of senile dementia,and it is also one of the diseases with the highest incidence in the elderly population.At present,the clinical treatment with acetylcholinesterase inhibitor(AChEI)or N-methyl-D-aspartate receptor antagonist(NMDA Antagonist)is still a palliative treatment,which can only moderately improve the cognition and behavior of patients with Alzheimer’s disease,but can not slow down the progression of the disease.Therefore,it is necessary to develop therapies aimed at the pathological mechanism of AD.Previous studies have shown that there is deposition of β-amyloid protein(Aβ),a pathological marker of AD,in the preclinical stage of cognitive impairment.Aβ is a product produced by the cleavage of amyloid precursor protein(APP)by Beta-site APP-Cleaving Enzyme 1(BACE1),which inhibits the excessive production of BACE1 and reduces the production of Aβ.It is an important therapeutic method to improve the cognition of AD and slow down the progression of the disease.Autophagy is the degradation and recovery of aging and damaged biological macromolecules and organelles by lysosomes,which plays an important role in cell development,differentiation,survival and maintenance of internal environment stability.Mitochondria are vital organelles to maintain cell energy metabolism,and the way of their metabolism is mitochondrial autophagy.The health of neurons depends on the elimination of A β and other toxic proteins by mitochondria by autophagy.Mitochondrial autophagy dysfunction runs through the pathogenesis and disease of AD.According to the theory of traditional Chinese medicine,the pathogenesis of AD is classified as deficiency and excess,deficiency of spleen and kidney for a long time to phlegm and blood stasis,treatment of tonifying drugs combined with promoting blood circulation and removing blood stasis,awakening brain resuscitation drugs and so on.Acorus tatarinowii is the representative medicine of Xingnao Kaiqiao medicine,which has the properties of aroma,awakening,awakening,low molecular weight,strong fat solubility and fast absorption in the body.β-asarone is one of the main effective components of Acorus calamus.In previous experiments,we found that β-asarone has a certain therapeutic effect on neurodegenerative diseases,nervous system tumors,depression,cerebral infarction and so on.Based on the BACE1 target,this paper studied the pharmacodynamic mechanism of β-asarone on APP/PS1 transgenic mice,and further studied its effects on autophagy and PINK 1/Parkin-mediated mitochondrial autophagy in APP/PS1 transgenic mice.This study is mainly composed of four parts of experiments.Part1:We assayed the effect of β-asarone on learning and memory ability and acetylcholine(ACh)metabolism in APP/PS1 transgenic mice,and to explore the effect of β-asarone on APP/PS1 transgenic mice.Part 2:We studied the effects of low,medium and high β-asarone on the deposition of BACE1,Aβ and the expression of APP,Presenilin-1,PS1 and neurosynaptophysin-1(Synapsin I,SYN-1)in the brain of APP/PS1 transgenic mice,and to evaluate its efficacy.Part3:We studied the effects of β-asarone on Aβdeposition and autophagy-related factors Beclin-1,p62 and LC3Ⅰ/Ⅱ in the brain of APP/PS1 transgenic mice,and to explore the effect of β-asarone on autophagy in APP/PS1 transgenic mice.Part 4:We observed the effect of β-asarone on mitochondrial autophagy mediated by PINK1/Parkin pathway in APP/PS1 transgenic mice,and to explore the possible mechanism and target of β-asarone on AD.Part 11.1 ObjectiveTo study the effect of β-asarone,the main active ingredient of Acorus tatarinowii on the learning and memory ability and acetylcholine(ACh)metabolism of APP/PS1 transgenic mice.1.2 Method50 SPF APP/PS1 transgenic mice,half female and half male,were randomly divided into 5 groups with 10 mice in normal group:model group,low-dose β-asarone group,medium-dose β-asarone group,high-dose β-asarone group and Donepezil group.In addition,there were 10 wild type mice with APP/PS1 genotype in normal group.Normal group and model group were given normal saline and intragastric administration for 30 days.①Behavioral experiments(water maze test,Passive avoidance test,step-down test)to evaluate the learning and memory ability of APP/PS1 transgenic mice;②ELIS A was used to detect the content of ACh in APP/PS1 transgenic mice.1.3 ResultsThe swimming time of the model group was significantly longer than the normal group,the number of errors was significantly increased(P<0.001),the latency of passive avoidance was significantly shortened(P<0.001),and the number of errors of passive avoidance was increased,the latency of step-down test was significantly shortened(P<0.001),the number of errors of step-down test showed an increasing trend,and the content of ACh decreased significantly(P<0.001)than the normal group.The swimming time of all treatment groupss decreased in the water maze test than the model group,and the swimming time of middle and high dose β-asarone groups decreased significantly(P<0.001),and the number of errors in treatment groups showed a downward trend.The latency of all treatment groupss was prolonged,and the latency of middle and high dose β-asarone groups was significantly longer than that of model group(P<0.05,P<0.001).The number of errors in treatment groups showed a downward trend,and the number of errors in high-dose β-asarone group decreased significantly(P<0.001).The number of errors in treatment groups showed a downward trend in the step-down test,and the number of errors in the low,middle and high dose of β-asarone group was significantly lower than that in the model group(P<0.01,P<0.05,P<0.05).The number of errors in Donepezil group was significantly lower than that in Donepezil group(P<0.01).Compared with the model group,the ACh in the cortex of mice in treatment groups increased significantly(P<0.05,P<0.001).Among them,the content of ACh in β-asarone low,middle and high dose groups increased significantly(P<0.001).1.4 Conclusionβ-asarone can improve the learning and memory ability of APP/PS1 transgenic mice,and its mechanism may be that it improves the metabolism of ACh in serum and cerebral cortex of APP/PS1 transgenic mice.Part 22.1 Object iveTo study the pharmacodynamic target of β-asarone in APP/PS1 transgenic mice based on BACE1.2.2 Method50 SPF APP/PS1 transgenic mice,half female and half male,were randomly divided into 5 groups with 10 mice in normal group:model group,low-dose β-asarone group,medium-dose β-asarone group,high-dose β-asarone group and Donepezil group.In addition,there were 10 wild type mice with APP/PS1 genotype in normal group.①ELISA detection of APP,PS1,Aβ,BACE1 and SYN-1 in the hippocampus of APP/PS1 transgenic mice;②RT-PCR detection of related genes:APP mRNA,PS1 mRNA,SYN-1 mRNA and BACE1 mRNA;③WB detection of BACE1 and SYN-1 in the hippocampus of APP/PS1 transgenic mice.2.3 Results2.3.1 ELISA detection of APP,PS1,Aβ,BACE1 and SYN-1 in the hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of APP,BACE1,Aβ and BACE1 in the hippocampus of the model group was significantly increased,while the expression of SYN-1 was significantly decreased,the difference was statistically significant(P<0.001),and the expression of PS1 showed an upward trend.Compared with the model group,the APP in the hippocampus of mice with high dose of β-asarone decreased significantly(P<0.001).The PS1 in the middle and high dose groups of β-asarone and Donepezil group decreased significantly(P<0.01,P<0.001).Aβin low,middle and high dose β-asarone groups and Donepezil group decreased significantly(P<0.01,P<0.001).The BACE1 of β-asarone low,middle and high dose groups and Donepezil group decreased significantly(P<0.05,P<0.001).The SYN-1 in the middle and high dose groups of β-asarone increased significantly(P<0.001).2.3.2 RT-PCR detection of related genes:APP mRNA,PS1 mRNA,SYN-1 mRNA and BACE1 mRNACompared with the normal group,the expression of APP mRNA,PS1 mRNA and BACE1 mRNA in the hippocampus of the model group increased significantly,while the expression of SYN-1 mRNA decreased significantly,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of APP mRNA in the hippocampus of mice in low,middle and high dose groups of β-asarone and Donepezil group decreased significantly(P<0.05-P<0.01).The expression of PS1 mRNA in the hippocampus of mice in low,middle and high dose groups of β-asarone and Donepezil group showed decreased significantly(P<0.05-P<0.01).The expression of BACE1 mRNA in hippocampus of mice in low,middle and high dose groups of β-asarone and Donepezil group decreased significantly(P<0.05-P<0.01).The expression of SYN-1 mRNA in the hippocampus of mice in the middle and high dose groups of β-asarone increased significantly(P<0.05-P<0.01).2.3.3 WB detection of BACE1 and SYN-1 in the hippocampus and cortex of APP/PS1 transgenic miceCompared with the normal group,the expression of BACE1 in the hippocampus of the model group increased and the level of SYN-1 decreased.Compared with the model group,the expression of BACE1 in the hippocampus of treatment groups decreased,and the level of SYN-1 in the high-dose β-asarone group increased(P<0.05).2.4 ConclusionDifferent doses of β-asarone could inhibit the expression of APP and PS1 in the hippocampus and cortex of APP/PS1 mice,and the high dose group(30mg/kg ·d)had the best effect,and could inhibit the expression of BACE1 and increase the expression of SYN-1 in the hippocampus of APP/PS1 transgenic mice.Part 33.1 Object iveTo study the effect of β-asarone on autophagy in APP/PS1 transgenic mice3.2 Method40 SPF APP/PS1 transgenic mice,half female and half male,were randomly divided into four groups:model group,moderate dose of β-asarone group,autophagy activator A(antimycin Acent A3)and inhibitor cyclosporine A(CyclosporinA,CsA)group.In addition,there were 10 wild type mice with APP/PS1 genotype in normal group.Normal group and model group were given normal saline and intragastric administration for 30 days.①WB detection of LC3I/II,Beclin-1 and p62 expression in the hippocampus and cortex of APP/PS1 transgenic mice;②RT-PCR detection of related genes:LC3 mRNA,Beclin-1 mRNA and p62 mRNA;③IHC detected Beclin-1,p62 and LC3 expression in the hippocampus of APP/PS1 mice.3.3 Results3.3.1 WB detection of β-asarone on the expression of LC3I/II,Beclin-1 and p62 in hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of LC3II/LC3I and Beclin-1 in the hippocampus of the model group decreased(P<0.01,P<0.05),while the expression of p62 increased significantly(P<0.01).Compared with the model group,the expression of LC3Ⅱ/LC3I in hippocampus of mice in β-asarone group was significantly increased(P<0.001),and the expression of LC3Ⅱ/LC3I in hippocampus of mice in A3 group was significantly increased(P<0.001).The expression of Beclin-1 in hippocampus of mice in β-asarone group increased,and the expression of Beclin-1 in hippocampus of mice in A3 group increased significantly(P<0.001).The expression of p62 in the hippocampus of mice in the β-asarone group decreased significantly(P<0.001),and the expression of p62 in the hippocampus of the A3 group decreased significantly(P<0.001).The expression of p62 in the hippocampus of mice in the),CsA group increased significantly(P<0.001).3.3.2.RT-PCR of β-asarone on the expression of Beclin-1 mRNA,p62 mRNA and LC3 mRNA in hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of LC3 mRNA and Beclin-1 mRNA in the hippocampus of the model group decreased(P<0.01),and the expression of p62 mRNA showed an upward trend.Compared with the model group,the expression of LC3 mRNA in the hippocampus ofβ-asarone mice and A3 group increased(P<0.001).The expression of Beclin-1 mRNA in the hippocampus of β-asarone mice and A3 group increased(P<0.05),CsA group decreased(P<0.05).The expression of p62 mRNA in the hippocampus of β-asarone group showed a downward trend,and the expression of p62 mRNA in the hippocampus of A3 group was significantly lower than that of the model group(P<0.01).The expression of p62 mRNA in the hippocampus of mice in the CsA group showed an upward trend.3.3.3.IHC of β-asarone on the expression of Beclin-1,p62 and LC3 in hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of LC3 and Beclin-1 in the hippocampus of the model group decreaed(P<0.05,P<0.001),the expression of p62 significantly increased(P<0.05).Compared with the model group,LC3 in the hippocampus of the β-asarone group and A3 group significantly increased(P<0.001).Compared with the model group,Beclin-1 in the hippocampus of the β-asarone group and A3 group significantly increased(P<0.001).Compared with the model group,p62 in the hippocampus of the β-asarone group and A3 group significantly decreased(P<0.05-P<0.01).3.4 ConclusionThe autophagy function of APP/PS1 transgenic mice was significantly inhibited than that of normal wild type mice.β-asarone can activate autophagy,promote the expression of LC3 and Beclin-1 and inhibit the expression of p62 in APP/PS1 transgenic mice.Part 44.1 Object i veTo study the effect of β-asarone on mitochondrial autophagy mediated by PINK1-Parkin pathway in APP/PS1 transgenic mice.4.2 Method40 SPF APP/PS1 transgenic mice,half female and half male,were randomly divided into 4 groups with 10 mice in each group,namely model group,medium dose β-asarone group,A3 group and CsA group.In addition,there were 10 wild type mice with APP/PS1 genotype in normal group.Normal group and model group were given normal saline and intragastric administration for 30 days.①RT-PCR detection of PINK1 mRNA and Parkin mRNA in hippocampus of APP/PS1 transgenic mice;②IHC detection of PINK 1 and Parkin in hippocampus of APP/PS1 transgenic mice.4.3 ResuIts4.3.1 RT-PCR detection of PINK1 mRNA and Parkin mRNA in hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of PINK1 mRNA in the hippocampus of the model group decreased(P<0.01),and Parkin mRNA showed an upward trend.Compared with the model group,the expression of PINK 1 mRNA in the hippocampus of mice in the β-asarone group and A3 group increased(P<0.01-P<0.001),and CsA group increased(P<0.01).The expression of Parkin mRNA in the hippocampus of β-asarone group and A3 group increased(P<0.05).The expression of Parkin mRNA in the hippocampus of CsA group showed an upward trend.4.3.2 IHC detection of PINK1 and Parkin in hippocampus of APP/PS1 transgenic miceCompared with the normal group,the expression of PINK1 and Parkin in the hippocampus of the model group decreased significantly(P<0.01).Compared with the model group,the expression of PINK1 in hippocampus of mice inβ-asarone group was significantly increased(P<0.001),and the expression of PINK1 in hippocampus of mice in A3 group was increased(P<0.001).The expression of Parkin in hippocampus of mice in β-asarone group increased significantly(P<0.001),and the expression of Parkin in hippocampus of A3 group increased significantly(P<0.05).4.4 ConclusionThe mitochondrial autophagy function of APP/PS1 transgenic mice was significantly inhibited than that of wild type mice.β-asarone activates PINK1/Parkin-mediated mitochondrial autophagy in APP/PS1 mice,which may be one of the mechanisms of the pharmacological effect of β-asarone on APP/PS1 mice.Final Conclusionβ-asarone can effectively improve the learning and memory ability of APP/PS1 transgenic mice,and its mechanism may be related to the improvement of ACh metabolism.Low,middle and high dose of β-asarone could inhibit the expression of APP,PS1 and BACE1 and increase the expression of SYN1 in the hippocampus of APP/PS1 transgenic mice,and the effect of high dose group(30mg/kg·d)was the most obvious.The autophagy function of APP/PS1 transgenic mice is significantly inhibited than that of normal mice.β-asarone can promote autophagy,increase the expression of LC3I/II and Beclin-1 and inhibit the expression of p62 in APP/PS1 mice,suggesting that β-asarone may play a role in promoting autophagy in APP/PS1 transgenic mice.β-asarone promotes PINK1/Parkin-mediated mitochondrial autophagy in APP/PS1 transgenic mice.