Study On The Effect And Mechanism Of β-asarone On AD Rats Based On PINK1/Parkin Mediated Mitophagy

Alzheimer’s disease(AD)is a chronic neurodegenerative disease,the prevalence of AD increased with age.The pathological features of AD include extracellular deposition of A β,neurofibrillary tangle predominant(NFT)and extensive neuronal loss.Many studies have shown that autophagy and the metabolism of amyloid precursor protein(APP),the formation of A β is closely related.As one well-studied type of selective autophagy,mitochondrial autophagy also involved in the pathogenesis of AD.Adenosine triphosphate(ATP)is an energy source necessary for normal life activities of cells,and mitochondria produce ATP to maintain cellular homeostasis and normal survival of neurons.In AD patients,the abnormal increase of Aβ affects the normal function of mitochondria,also hinders the self-clearance of damaged mitochondria.Mitochondrial dysfunction plays an important role in the pathogenesis of AD.In traditional Chinese medical theory,the pathogenesis of AD mainly based on qi deficiency,obstruction by phlegm,and blood stasis.In terms of treatment,the resuscitation-inducing herbs represented by Acorus gramineus,borneol,muscone and styrax have the fragrance of opening the orifices and arousing the brain channeling.Resuscitation-inducing herbs are widely used in ischemic encephalopathy and AD.The effective components of resuscitation-inducing herbs generally have low molecular weight,which can regulate the permeability of the blood brain barrier and promote other pharmaceutical to cross the blood brain barrier.The common action characteristics of resuscitation-inducing herbs are fast absorption,wide distribution,and strong liposoluble.β-Asarone is one of the major effective components in Acorus calamus.Previous experiments suggested that β-asarone had certain therapeutic effects for neurodegenerative diseases,neurological tumors and depression,cerebral infarction.This article investigated the general character of four resuscitation-inducing herbs acted on AD rats in experiment part 1,then we selected β-asarone for further research to observed its effect on the autophagy function,PINK1/Parkin mediated mitophagy of AD rats.This study is mainly composed of four parts of experiments.Part 1:We assayed the learning and memory ability of AD rats by water maze test,we evaluated AchE level in rats brain,ET,CGRP level in rats serum by ELISA to explore the effects of four resuscitation-inducing herbs on AD rats induced by Aβ1-42 intracerebral injection.Part 2:It was observed in the result of part 1 that after pharmaceutical treatment to all groups of AD rats,β-asarone group showed better pharmacodynamic effects.Therefore,We evaluated the APP,PS1,BACE1,SYN-1 expression in AD rats brain by WB and PCR,to further explored the effects of different doses of β-asarone on AD rats.Part 3:We studied the effect of β-asarone on autophagy-related factors Beclin-1,p62,LC3I/II in the hippocampal tissue of AD rats by WB and PCR,to explore the effect of β-asarone on autophagy function in AD model rats.Part 4:We observed the effect of β-asarone on the mitochondrial autophagy mediated by the PINK1/Parkin pathway in AD rats,and explored the possible mechanism and targets of β-asarone acting on AD rats.Part 11.1 ObjectiveIn this research,we used the major active ingredients of four resuscitation-inducing herbs,β-asarone,borneol,styrax,and muscone,to observe the pharmacodynamic actions of resuscitation-inducing herbs on AD rats.1.2 Method90 SPF grade SD rats,half male and female,except for the blank group,other animal models were established by bilateral injection of Aβ1-42 in the hippocampus.We used water maze test to screen successful modeling AD rats.Rats were randomly divided into 7 groups including control group,model group,β-asarone group,borneol group,muscone group,storax group and donepezil group.The control group and the model group were given normal saline and administered by intragastric for 30 days.①Water maze test to evaluate the learning and memory ability of AD rats;②ELISA to detect the level of CGRP,ET,AchE in AD rats.1.3 ResultsThe escape latency of the model group rats in the water maze test prolonged than the control group(P<0.05),the expressions of ET and AchE significantly increased(P<0.05/P<0.01),and the expression of CGRP reduced than the control group(P<0.01).The escape latency of AD rats in each medication group shortened in the water maze test than the model group(P<0.05/P<0.01,P<0.01/P<0.01).β-asarone group showed a better effect among four resuscitation-inducing herb groups(P<0.001).The expression of ET and AchE in cortex of AD rats in each medication group decreased significantly(P<0.01/P<0.001/P<0.05/P<0.01),and CGRP increased significantly than the model group(P<0.05/P<0.05/P<0.01/P<0.01).1.4 Conclusionβ-asarone,borneol,storax and muscone improved the learning and memory ability of AD rats,and the mechanism may be that four above-mentioned resuscitation-inducing herbs improved AchE metabolism in the cerebral cortex of AD rats.β-asarone,borneol,storax and muscone improved the blood circulation of brain tissue by reduced the serum ET level and increased CGRP level of AD rats,there were some similarities between the intervention effects of the four resuscitation-inducing herbs on AD model rats.β-asarone,borneol,storax and muscone have certain pharmacodynamic effects on AD rats,and β-asarone had a better effect.Part 22.1 ObjectiveTo study the effect of different doses β-asarone on AD rats.2.2 Method80 SPF grade SD rats,half male and female,except for the blank group,the other animal models were established by bilateral injection of Aβ1-42 in the hippocampus.We used water maze test to screen successful modeling AD rats.Rats were randomly divided into 6 groups including control group,model group,β-asarone low-dose group(15mg/kg·d),β-asarone medium-dose group(30mg/kg·d),β-asarone high-dose group(45mg/kg·d)and donepezil group.The control group and the model group were given normal saline and administered by intragastric for 30 days.① WB detection of APP,PS1,SYN-1,BACE1 expression in the hippocampus of AD rats;② RT-PCR detection of related genes:APP mRNA,PS1 mRNA,SYN-1 mRNA,BACE1 mRNA.2.3 Results2.3.1 WB detection of β-asarone on the expression of APP,PS1,BACE1,and SYN-1 in the hippocampus of AD ratsThe expressions of APP,PS1,and BACE1 in the model group were significantly increased than control group(P<0.001),while the expression of SYN-1 was significantly decreased than control group(P<0.001).The expression of PS1 in the β-asarone low-dose group and donepezil group decreased than model group(P<0.01),the expression of PS1 in the β-asarone medium-dose group and β-asarone high-dose group significantly reduced than model group(P<0.001).The expression of SYN-1 in the β-asarone high-dose group and donepezil group increased than model group(P<0.05/P<0.001).The expression of BACE1 in the β-asarone high-dose group reduced than model group(P<0.05).2.3.2 RT-PCR to detect the effect of β-asarone on the expression of APP mRNA,PS1 mRNA,BACE1 mRNA and SYN-1 mRNA in the hippocampus of AD ratsThe expressions of APP mRNA,PS1 mRNA and BACE1 mRNA in the model group significantly increased than control group(P<0.001).The expressions of APP mRNA in the β-asarone low-dose group and β-asarone medium-dose group decreased than model group(P<0.05),in the β-asarone high-dose group and donepezil group decreased than model group(P<0.01).The expressions of PS1 mRNA in the β-asarone medium-dose group decreased than model group(P<0.05),in the β-asarone high-dose group and donepezil group decreased than model group(P<0.01/P<0.001).The expression of BACE1 in the β-asarone high-dose group reduced than model group(P<0.01).2.4 ConclusionDifferent dose β-asarone inhibited the expression of APP and PS1 in the hippocampus of AD rats,the high-dose β-asarone(45mg/kg·d)group was more effective,and showed the effect of inhibiting BACE1 and increasing the expression of SYN-1.Part 33.1 ObjectiveTo study the effect of β-asarone on autophagy in AD model rats.3.2 Method70 SPF grade SD rats,half male and female,except for the blank group,other animal models were established by bilateral injection of Aβ1-42 in the hippocampus.We used water maze test to screen successful modeling AD rats.Rats were randomly divided into 5 groups including control group,model group,β-asarone group,3-MA group and rapamycin group.The control group and the model group were given normal saline and administered by intragastric for 30 days.① WB detection of LC3II、Beclin-1、p62 expression in the hippocampus of AD rats;② RT-PCR detection of related genes:LC3 mRNA、Beclin-lmRNA、p62 mRNA;③ IHC detected Beclin-1 and p62 expression in thehippocampus of AD rats.3.3 Results3.3.1 WB detection of β-asarone on the expression of LC3II,Beclin-1,p62 in the hippocampus of AD ratsThe expression of LC3II and Beclin-1 in the model group decreased than the control group(P<0.05/P<0.001),and the expression of p62 increased than the control group(P<0.05).The expressions of LC3II and Beclin-1 in the β-asarone group increased than the model group(P<0.05/P<0.01);The expressions of LC3II,Beclin-1 decreased in 3-MA group(P<0.01/P<0.001),increased in rapamycin group than model group(P<0.01/P<0.01),the expression of p62 decreased in rapamycin group than the model group(P<0.001).3.3.2 RT-PCR to detect the effect of β-asarone on the expression of Beclin-1 mRNA,p62 mRNA,LC3 mRNA in the hippocampus of AD ratsThe expressions of Beclin-1 and LC3 in the model group decreased(P<0.001/P<0.05),and the expression of p62 increased than the control group(P<0.01).The expressions of Beclin-1 and LC3 in the β-asarone group increased(P<0.01/P<0.05),and the expression of p62 decreased than the model group(P<0.05).The expressions of Beclin-1 and LC3 in the rapamycin group increased(P<0.01/P<0.05),and the expression of p62 decreased(P<0.01);the expression of p62 in the 3-MA group increased than the model group(P<0.05).3.3.3 IHC detects the effect of β-asarone on the expression of Beclin-1 and p62 in the hippocampus of AD ratsThe expression of Beclin-1 in the model group reduced(P<0.001),and the expression of p62 increased than the control group(P<0.05).The expressions of Beclin-1 in the β-asarone group and donepezil group increased(P<0.05/P<0.05);p62 expression decreased than the model group(P<0.05/P<0.05).3.4 ConclusionThe autophagy function of AD rats is obviously inhibited than normal rats.β-Asarone activated autophagy in AD rats,promoted the expression of LC3,Beclin-1,and inhibited the expression of p62.Part 44.1 ObjectiveTo study the effect of β-asarone on the PINK1/Parkin pathway mediated mitophagy in AD rats.4.2 Method50 SPF grade SD rats,half male and female,except for the blank group,other animal models were established by bilateral injection of Aβ1-42 in the hippocampus.We used water maze test to screen successful modeling AD rats.Rats were randomly divided into 3 groups including model group,β-asarone group,CsA group.The control group and the model group were given normal saline and administered by intragastric for 30 days.① WB detection of PINK1,Parkin、β-Parkin expression in the hippocampus of AD rats;②RT-PCR detection of related genes:Parkin mRNA;③IHC detected PINK1 and Parkin expression in the hippocampus of AD rats.4.3 Results4.3.1 WB detection of β-asarone on the expression of PINK1,Parkin,and β-Parkin in the hippocampus of AD ratsThe expression of PINK1,Parkin,and β-Parkin in the model group all decreased than the control group(P<0.01/P<0.05/P<0.05).The expression of PINK1 and β-Parkin in the β-asarone group increased(P<0.001/P<0.001)than the model group.4.3.2 RT-PCR to detect the effect of β-asarone on the expression of Parkin mRNA in the hippocampus of AD ratsParkin mRNA expression in the model group decreased than the control group(P<0.01).Parkin mRNA expression in the β-asarone group increased than the model group(P<0.01).4.3.3 IHC detects the effect of β-asarone on the expression of PINK1 and Parkin in the hippocampus of AD ratsThe expressions of PINK 1 in the model group and the CsA group decreased(P<0.001/P<0.05);the expressions of Parkin decreased than the control group(P<0.001/P<0.01).The expressions of PINK1 and Parkin in the β-asarone group increased than the control group(P<0.05/P<0.05).4.4 ConclusionAD model rats’ mitochondrial autophagy function was significantly inhibited than normal rats.β-Asarone activated mitochondrial autophagy,and may exerted a pharmacodynamic effect on AD rats through the PINK1/Parkin pathway.Final ConclusionThe four resuscitation-inducing herbs such as Acorus gramineus,bomeol,muscone,styrax improved the learning and memory ability of AD rats.β-Asarone had a better effect,the mechanism may be related to β-asarone improved brain blood circulation and Ach metabolism.Low,medium and high doses β-asarone inhibited the expression of APP and PS1,and protected the neurosynaptic function of AD rats.The high-dose β-asarone group(45mg/kg.d)had a better effect,while high dose β-asarone inhibited BACE1 and increased the expression of SYN-1.The autophagy function of AD model rats inhibited compared with normal rats.β-asarone promoted autophagy function in AD rats,increased the expression of LC3II,Beclin-1,and inhibited p62,which suggests that β-asarone may exerted therapeutic effects by promoting autophagy in AD rats.β-Asarone promotes PINK 1/Parkin-mediated mitochondrial autophagy in AD rats..