| ?-asarone,the main effective component extracted from Acorus tatarinowii Schott,it plays a significant protective effect on the central nervous system.Previous studies demonstrated that ?-asarone could improve behavior ability of 6-OHDA induced Parkinson's rat via JNK/Bcl-2/Beclin-1 pathway.Autophagy is a cellular process to degrade long-lived proteins and recycle cellular components,which plays an important role in PD treatment.However,whether?-asarone and levodopa co-administration have the role of increasing striatal DA level via affecting the related enzymes of DA in Parkinson's disease model?The duration of ?-asarone and levodopa(L-dopa)co-administration in Parkinson's rats is how long?What is mechanism of ?-asarone and L-dopa co-administration?Whether ?-asarone and L-dopa co-administration could improve the symptoms of Parkinson' s disease via modulating autophagy pathway?The experiment was mainly composed of six parts,part ?:To investigate whether the L-dopa combined with ?-asarone could increase dopamine(DA)of the brain and its mechanism;Part ?:to investigate the dynamic changes of L-dopa,dopamine(DA),3,4-Dihydroxyphenylacetic acid(DOPAC),homovanillic acid(HVA),5-hydroxytryptamine(5-HT),Tyrosine hydroxylase(TH)and catechol-Omethyltransferase(COMT)in rat plasma and brain tissue(striatum,cortex and hippocampus)during 48h after co-administration of ?-asarone and L-dopa;Park ?:To explore the effects and mechanisms of co-administration in PD rats,behavioral tests,neurotransmitters,Dopa decarboxylase(DDC),tyrosine hydroxylase(TH),catechol-0-methyltransferase(COMT)and monoamine oxidase B(MAO-B)levels were detected;Park ?:To investigate the effects of ?-asarone and L-dopa co-administration on BBB and P-glycoprotein(P-gp);Park V:To investigate Beclin-1,light chain 3B(LC3B)and p62 expression in 6-hydroxydopamine(6-OHDA)-induced parkinsonian rats after 0-asarone and L-dopa coadministration;Park VI:To explore the mechanism of ?-asarone and L-dopa coadministration affecting on MEF2D and a-syn via regulating HSP70/MAPK/MEF2D/Beclin-1 pathway in 6-hydroxydopamine(6-OHDA)-induced parkinsonian rats.1.1 ObjectiveThe aim is to study the effect of ?-asarone and L-dopa co-administration on monoamine neurotransmitters level in the striatum of healthy rats.1.2 MethodsRats were randomly divided into four groups(n=10):normal group(normal saline),madopar group(75mg/kg,L-dopa:Benserazide=4:1),L-dopa group(60mg/kg,L-dopa)and co-administered group(15mg/kg,?-asarone;60mg/kg,L-dopa).All rats were given by intragastric administration twice a day for 7 days.Then DA,L-dopa,serotonin(5-HT),homovanillic acid(HVA)and 3,4-Dihydroxyphenylacetic acid(DOPAC),tyrosine hydroxylase(TH),catecholamine oxygen methyl transferase(COMT)and monoamine oxidase B(MAO-B)in plasma and striatum were determined.Striatum was observed by hematoxylin and eosin(HE)staining.1.3 Results1.3.1 The changes of neurotransmitters in the striatumStriatal level of L-dopa,DA,HVA and 5-HT increased significantly in madopar,L-dopa and co-administered groups compared with that of normal group(P<0.05).DOPAC in the madopar and co-administered groups also increased compared with that of normal group(P<0.05).In addition,L-dopa and HVA increased significantly in madopar group;DOPAC also increased significantly in co-administered group compared with that of L-dopa group(P<0.01).As compared to madopar group,L-dopa decreased significantly in the co-administered group(P<0.01)and DOPAC increased significantly in the co-administered group(P<0.01).1.3.2 The changes of neurotransmitters in plasmaAs compared to normal group,L-dopa,DA,DOPAC and HVA increased significantly in the madopar,L-dopa and co-administered groups(P<0.01).5-HT showed no significant difference in each groups.Compared with L-dopa group,DA decreased in the madopar group(P<0.05);DA,DOPAC and HVA increased significantly in the co-administered group(P<0.05).As compared to madopar group,DA,DOPAC and HVA increased significantly in co-administered group(P<0.01).1.3.3 Pathologic conditionCompared to normal group,the number of neurons increased in the madopar,L-dopa and co-administered groups.1.4 ConclusionsIn conclusion,?-asarone and L-dopa co-administration not only can increase L-dopa,DA,DOPAC and HVA in the striatum and plasma,but also can protect dopaminergic neurons in the striatum.2.1 ObjectiveThe aim of the study is to investigate the dynamic changes of monoamine neurotransmitters in rat plasma and brain tissue(striatum,cortex and hippocampus)during 48h after co-administration of ?-asarone and L-dopa.2.2 MethodsIn this study,rats were administrated ?-asarone and L-dopa(at the dose of 60 and 15mg/kg body weight,respectively)at a time by intragastric.After that,rats were randomly assigned into five groups(n=4):control(0h)group,1h group,5h group,18h group and 48h group according to the sacrifice time.Then DA,L-dopa,DOPAC,HVA and 5-HT in plasma,striatum,cortex and hippocampus were determined by HPLC.2.3 Results2.3.1 The changes of neurotransmitters in the brain and plasmaIn striatum,L-dopa increased at 0 to 1h,peaking at 1h,and decreased'dramatically between 1 and 5h,then remained steady among 5,18 and 48h in striatum.The peak value of DA was at 1h,it kept steady between 1 and 5h and showed slightly decreased from 5 to 18h,however,it increased during 18-48h.DOPAC was increased within 1h,peaking at 1h,but dropped from 1 to 5h.The amount of HVA in striatum were peaked at 1h,while it declined dramatically between 1 and 5h,then it remained steady from 5 to 48h.Besides,5-HT kept steady during 48h.In the cortex,hippocampus and plasma,L-dopa,DA,DOPAC and HVA showed a very similar trend,they peaked at 1h and kept steady from 5 to 48h.And 5-HT kept steady from Oh to 48h.2.3.2 The dynamic changes of the related enzymes2.3.2.1 In striatumTH levels in striatum increased at 1h and returned to normal level from 5 to 48h.Dopa decarboxylase(DDC)levels remained rose during 48h and peaked at 5h.COMT levels were below the normal level during 48h and kept steady.MAO-B levels were also below the normal level during 48h and remained a slowly trend.2.3.2.2 In plasmaTH levels increased significantly at 1h and returned to normal level from 5h to 48h.DDC levels increased significantly during 48h and close to normal level at 5h.COMT level was below normal level during 48h and kept a slowly reduction.2.4 Conclusions?-asarone and L-dopa co-administration increased DA levels in striatum and plasma by enhancing DDC activity.DA in striatum remained a higher level during 48h.DA metabolism decreased in striatum by inhibiting COMT and MAO-B activities.But DA metabolism in plasma was enhanced.3.1 ObjectiveThe aim of the study is to investigate the effects and mechanisms of?-asarone and L-dopa co-administration on monoamine neurotransmitters in PD rats.3.2 MethodsSix ? L 6-OHDA was then injected into each rat at the following coordinates of Paxinos and Watson(1982):tooth bar:-2.3 mm,antero-posterior:-4.4 mm,medio-lateral:-1.2 mm,dorso-ventral:-7.8 mm(Paxinos and Watson 1982).The injection speed was 0.5 ?L/min,and after that the needle was left for 15 min.The sham-operated rats(n=10,group A)were injected with equivalent amount of 0.9%(w/v)NaCl containing 0.02%L-ascorbic acid into the MFB as the above described stereotaxic coordinates.One month after the injection,seventy PD rats were successfully chosen according to apomorphine(0.5 mg/kg,i.p.Sigma,USA)ratation test(7 circles/min).The PD rats were randomly assigned into the seven groups(n=10):group B(model),group C(madopar,75 mg/kg),group D(L-dopa,60 mg/kg),group E(0-asarone,15 mg/kg),group F(low dose of ?-asarone + L-dopa:7.5+60 mg/kg),group G(medium dose of?-asarone+L-dopa:15+60 mg/kg),and group H(high dose of ?-asarone+L-dopa:30+ 60 mg/kg).All the treated-rats were given intragastric administration(5 mL/kg)twice a day for 30 days consecutively.Then behavioral tests and neurotransmitters in striatum and plasma were determined.Dopa decarboxylase(DDC),tyrosine hydroxylase(TH),catechol-0-methyltransferase(COMT)and monoamine oxidase B(MAO-B)were detected.Besides,the pathological characteristics of liver and kidney and ultrastructure of dopaminergic neurons were observed.3.3 Results3.3.1 Behavioral testsIn open-field test,the number of moving squares,the standing number of hindlimb and feces number increased significantly in each dose of co-administered group(P<0.05)compared with that of model group;furthermore,the time spent in the central square of the arena in low dose of co-administered group shortened comparably to model group(P<0.05).Compared with madopar and L-dopa groups,the number of moving squares showed a significant increase in high dose of co-administered group(P<0.05),and the standing number of hindlimb exhibited no significant difference between medium and high dose of co-administered groups.The feces number in high dose of co-administered group was more than that of L-dopa group(P<0.05),however,it was not significantly different compared with that of madopar group.The time spent in the central square of the arena in high dose of co-administered group was less than that of madopar and L-dopa groups(P<0.05).In rotarod test,the time that rats stayed on the rotarod in high dose of co-administered group was prolonged compared with that of L-dopa group(P<0.05),but it manifested no significant difference compared with that of madopar group.In stepping test,the initiation time of the right forelimb reduced(P<0.05)in madopar,L-dopa and each dose of co-administered groups,the stepping time of the right forelimb shortened in each dose of co-administered group(P<0.05),and the number of steps of the right forelimb increased in all treated-groups(P<0.05)compared with that of model group.Compared with L-dopa group,the initiation time and the stepping time of the right forelimb were less than that of high dose of co-administered group(P<0.05);in contrast,the number of steps of the right forelimb in high dose of co-administered group enhanced(P<0.05).However,all of these in co-administered groups had no significant difference by comparable to that of madopar group.3.3.2 The changes of neurotransmitters in the striatumThere was no significant difference in L-dopa levels between ?-asarone and L-dopa groups.Our results showed that L-dopa levels in madopar group were far higher than that of each dose of co-administered group.DA levels increased significantly in madopar,L-dopa and each dose of co-administered groups(P<0.05)compared with that of model group.What was interesting was that DA levels in each dose of co-administered group were similar to that of madopar group.DOPAC,HVA and 5-HT levels augmented significantly in madopar,L-dopa,?-asarone and each dose of co-administered groups.L-dopa levels in each dose of co-administered groups enhanced significantly compared with that of madopar and L-dopa groups(P<0.01).Meanwhile,DA,DOPAC,HVA and 5-HT levels in high dose of co-administered group also increased significantly compared with that of L-dopa group(P<0.05),but they showed no significant difference compared with that of madopar group.3.3.3 The changes of neurotransmitters in plasmaL-dopa,DA,DOPAC,HVA and 5-HT levels increased significantly in madopar,L-dopa,?-asarone and each dose of co-administered groups(P<0.05)compared with that of model group.Compared with madopar and L-dopa groups,L-dopa levels in each dose of co-administered groups decreased significantly(P<0.05).And the DOPAC and HVA levels in low and medium dose of co-administered group were lower than that of madopar and L-dopa groups(P<0.05).In contrast,DA and 5-HT levels in medium and high dose of co-administered groups were higher than that of L-dopa group(P<0.05),but they displayed no significant difference compared with that of madopar group.3.3.4 Biochemical analysisCompared with AST and ALT levels in model group,AST decreased in madopar group(P<0.01)and ALT increased significantly in L-dopa group(P<0.01),and AST and ALT levels had no significant difference among each dose of co-administered group(P>0.05).In addition,compared with the CR levels in model group,the CR levels reduced significantly in madopar and each dose of co-administered groups(P<0.05),but they enhanced significantly in L-dopa group.Compared with BUN and total bilirubin levels in model group,BUN declined(P<0.01),whereas total bilirubin showed no significant difference in all treated-groups.Besides,compared with AST,BUN,ALT and CR levels in L-dopa group,AST and BUN diminished significantly in high dose of co-administered group(P<0.05);ALT dropped significantly in each dose of co-administered groups(P<0.05);CR lessened in both medium and high dose of co-administered(P<0.01).Compared with AST,BUN,ALT and CR levels in madopar group,they showed no significant difference in each dose of co-administered group.3.3.5 The dynamic changes of the related enzymesIn striatum,MAO-B,COMT,TH and DDC levels increased but DAT levels decreased significantly in madopar,L-dopa,?-asarone and each dose of co-administered groups compared with that of model group(P<0.05).In addition,compared with L-dopa group,MAO-B,COMT and TH levels increased excepting that DAT levels declined significantly in medium and high dose of co-administered groups(P<0.05).Furthermore,MAO-B,COMT and TH levels decreased significantly in low dose of co-administered groups compared with that of madopar group(P<0.05).In plasma,DDC levels declined significantly in madopar and L-dopa groups,but it increased significantly in ?-asarone and each dose of co-administered groups(P<0.05);TH and COMT levels in madopar group,L-dopa,?-asarone and each dose of co-administered groups were higher than that of model group(P<0.05).In addition,DDC levels augmented significantly in high dose of co-administered group compared with that of L-dopa and madopar groups(P<0.05).COMT and TH levels in medium and high dose of co-administered groups increased compared with that of L-dopa group(P<0.05),however,they showed no significant difference compared with that of madopar group.3.3.6 Liver and kidney histopathologyThe histopathology examination of liver tissue sections in all treated groups also revealed no structural changes compared with that of sham-operated group.And the histopathology study of the kidney tissues in L-dopa group showed glomerulus atrophy,inflammatory cells infiltration into renal interstitium,hinting L-dopa could induce kidney damage.3.3.7 TH immunohistochemistryThe expression of TH positive neurons increased significantly in madopar,L-dopa,?-asarone and each dose of co-administered groups compared with that of model group(P<0.05).Moreover,the expression of TH positive neurons in each dose of co-administered group were higher than that of L-dopa group(P<0.05),however,TH in high dose of co-administered group displayed no significant difference compared with that of madopar group.3.3.8 The ultrastructure of dopaminergic neuronsThe dopaminergic neurons in sham-operated group were normal.Smaller cell body,expanded rough endoplasmic reticulum,increased lysosome,swollen and vacuolated mitochondria were found.Besides,karyopyknosis and chromatin gathered along the nuclear membrane in model group,which were the ultrastructural characteristics of autophagy.However,the cell body became normal with clear nucleolus,the amount of lysosome and mitochondria decreased in each dose of co-administered group.3.3.9 The changes of DDC levelsImmunofluorescent analyses both manifested that striatal DDC levels in madopar,L-dopa,?-asarone and each dose of co-administered groups were obviously higher than that of model group(P<0.05).Furthermore,striatal DDC levels in ?-asarone and each dose of co-administered groups increased significantly compared with that of madopar and L-dopa groups(P<0.05).However,striatal DDC levels showed no significant difference between L-dopa and madopar groups(P>0.05).3.4 Conclusions?-asarone and L-dopa co-administration can improve the behavioral abilities and augment striatal DA levels via enhancing DDC and TH activities.And it also could alleviate the damage of dopaminergic neurons induced by 6-OHDA and protect liver and kidney of PD rats.4.1 ObjectiveThe aim of the study is to investigate the effects of ?-asarone and L-dopa co-administration on BBB and P-glycoprotein(P-gp)in 6-OHDA induced PD rats.4.2 MethodsUnilateral 6 ? L 6-OHDA(4 mg/mL,Sigma,free base dissolved in a solution of 20 mg/mL L-ascorbic acid in 0.9%w/v normal saline)injection into the left medial forebrain bundle was used in rats at following coordinates according to the coordinates of Paxinos and Watson(1982)(flat scull,as above):tooth bar:-2.3mm,antero-posterior:-4.4 mm,medio-lateral:-1.2 mm,dorso-ventral:-7.8 mm(Paxinos and Watson,1982).The injection speed was 0.5 u L/min,and after that the needle was left for 15 min.The sham rats(n=10)received the same volume of vehicle according to the same procedure.One month after the injection,56 PD rats were successfully established as judged by contralateral rotations induced by apomorphine(0.05 mg/kg,i.p.Sigma,USA).The PD rats were divided into four groups(n=14):model group,L-dopa group,?-asarone group,L-dopa+?-asarone group.All the treated-rats were given by intragastric administration(5 mL/kg)twice a day for consecutive 30 days.Then striatal L-dopa,DA,3,4-Dihydroxyphenylacetic acid(DOPAC)and homovanillic acid(HVA)levels were determined.P-gp,zonula occludens-1(Z0-1),claudin-5,occludin and actin expressions in prefrontal cortex were detected.4.3 Results4.3.1 The changes of L-dopa,DA,DOPAC and HVA levels in the striatumL-dopa,DA,DOPAC and HVA levels decreased significantly in model group(P<0.05)compared with those of sham group.L-dopa,DA,DOPAC and HVA levels in co-administered group and L-dopa group increased significantly(P<0.05)compared with those of model group.Importantly,L-dopa and DA levels showed a significant increase in co-administered group compared with that of L-dopa group(P<0.05).DOPAC and HVA levels in co-administered group were lower than that of L-dopa group(P<0.05).4,3.2 The changes of P-gp,ZO-1,occludin,actin levels in prefrontal cortexThe expression levels of P-gp,ZO-1,occludin,actin decreased significantly in model group compared with those of sham group(P<0.05).Meanwhile,the expression levels of P-gp,ZO-1,occludin,actin decreased significantly between ?-asarone and co-administered groups compared with those of model and L-dopa groups(P<0.05).4.3.3 Results of P-gp expression level by immunohistochemistry staining analysesThe expression levels of P-gp decreased in model group compared with that in sham group(P<0.05).Meanwhile,the expression levels of P-gp also decreased significantly between ?-asarone and co-administered groups compared with those in model group(P<0.05).4.3.4 Results of ZO-1 expression level by immunofluorescent assayThe expression levels of ZO-1 decreased in model group compared with that in sham group(P<0.01).Meanwhile,the expression levels of ZO-1 also decreased significantly between ?-asarone and co-administered groups compared with those in model group(P<0.01).4.3.5 Results of claudin-5 expression levels by immunohistochemistry stainingThe expression levels of claudin-5 decreased in model group compared with that in sham group(P<0.05).Meanwhile,the expression levels of claudin-5 also decreased significantly between 13-asarone and co-administered groups compared with those in model group(P<0.05).4.4 Conclusions?-asarone and L-dopa co-administration could increase striatal levels of L-dopa and DA in PD rats by down-regulating P-gp,ZO-1,claudin-5,occludin and actin expressions in prefrontal cortex.5.1 ObjectiveThe aim of the study is to investigate Beclin-1,light chain 3B(LC3B)and p62 expression in 6-hydroxydopamine(6-OHDA)-induced parkinsonian rats after ?-asarone and levodopa(L-dopa)coadministration.5.2 MethodsSevere unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?1 6-OHDA(4?g/?l,Sigma,free based dissolved in a solution of 20 mg/ml L-ascorbic acid in 0.9%normal saline)at a rate of 0.2 ?l/min into the medial forebrain bundle(MFB)at following coordinates according to the coordinates of Paxinos and Watson(1982)(flat scull,as above):tooth bar:-2.3 mm,anteroposterior:-4.4 mm,medio-lateral:1.2 mm,dorso-ventral:-7.8 mm.The rats in the sham-operated group received the same volume of normal saline according to the same procedure except for the 6-OHDA lesions.Rats were divided into eight groups:sham-operated and model groups;madopar group,administered madopar(75 mg/kg,p.o.);L-dopa group,administered L-dopa(60 mg/kg,p.o.);?-asarone group,administered?-asarone(15 mg/kg,p.o.);coadministered group,administered ?-asarone+ L-dopa(15mg/kg+60mg/kg,p.o.);3-Methyl adenine(3-MA)group,administered 3-MA(500 nmol,i.p.);rapamycin group,administered rapamycin(1 mg/kg,i.p.).Then Beclin-1,LC3B and p62 expression in mesencephalon were detected.The mesencephalon was also observed by transmission electron microscope.5.3 ResultsThe results showed that Beclin-1 and LC3B expression decreased(P<0.01)and p62 expression increased significantly(P<0.01)in madopar,L-dopa,?-asarone and coadministered treated groups compared with that of 6-OHDA model group.Among them,Beclin-1 and LC3B expression in ?-asarone and coadministered treated groups were less than that of madopar or L-dopa treated groups(P<0.05),in contrast,p62 expression in ?-asarone and coadministered treated groups was higher than that of madopar or L-dopa treated groups(P<0.05).In addition,a significant decrease in autophagosome was exhibited in ?-asarone and coadministrered treated groups compared with 6-OHDA model group(P<0.05).5.4 ConclusionThe coadministration of ?-asarone and L-dopa could decrease Beclin-1 and LC3B expression and increase p62 expression.6.1 ObjectiveThe aim of the study is to investigate the mechanism of ?-asarone and L-dopa coadministration affecting on MEF2D and ?-syn via regulating HSP70/MAPK/MEF2D/Beclin-1 pathway.6.2 Methods6-OHDA(Sigma,USA)was dissolved in the solution of 0.02%L-ascorbic acid and 0.9%(w/v)normal saline,which had a final concentration of 4 mg/mL.It diluted as the free base.6 u L 6-OHDA was then injected into each rat at the following coordinates of Paxinos and Watson(1982):tooth bar:-2.3 mm,antero-posterior:-4.4 mm,medio-lateral:-1.2 mm,dorso-ventral:-7.8 mm.The injection speed was 0.5 ?L/min,and after that the needle was left for 15 min.One month after the injection,sixty PD rats were successfully choosed according to apomorphine(0.05 mg/kg,i.p.Sigma,USA)ratation test(no less than 7 circles/min in 30 min).The sham-operated rats(n=10)were injected with equivalent amount of 0.9%(w/v)normal saline containing 0.02%L-ascorbic acid into the MFB as the above described stereotaxic coordinates.The 6-OHDA-lesion rats were randomly assigned into the five groups(n=12):6-OHDA-lesion model group,SB203580-treated group(SB203580,0.5mg/kg,i.p.),LiCl-treated group(LiCl,100mg/kg,i.p.),?-asarone-treated group(?-asarone,15 mg/kg,p.o.),coadministered-treated group(?-asarone 15 mg/kg + L-dopa 60 mg/kg,p.o.).All the treated-rats were given by intragastric administration or intraperitoneal injection(0.5 mL/kg)twice a day for consecutive 30 days.Tyrosine hydroxylase(TH),?-syn,lysosome-associated membrane protein type 2a(LAMP-2A),MEF2D,heat-shock protein 70(HSP70),Beclin-1,LC3B and p62 levels in mesencephalon were investigated.6.3 ResultsResults showed that ?-syn,Beclin-1 and LC3B levels increased but TH,LAMP-2A,p62 expressions decreased in model group.After ?-asarone or?-asarone plus L-dopa treatment,?-syn reduced but HSP70,MEF2D and TH levels enhanced,which suggested that the damage of dopaminergic neurons were attenuated;LAMP-2A levels augmented,which indicated that CMA was stimulated;Beclin-1,LC3B downregulation and p62 upregulation,which suggested that macroautophagy activity was inhibited.6.4 ConclusionThe co-administration could activate MEF2D activity and reduce ?-syn aggregation via activating CMA,inhibiting macroautophagy and upregulating HSP70 expression.Final ConclusionsThe coadministration of ?-asarone and L-dopa could increase L-dopa,DA,DOPAC and HVA levels in the striatum and plasma of healthy rats and PD model rats.In addition,the livers and kidneys of PD rats did not present any structural and pathological changes after the co-administration treatment.The co-administration also could elevate the BBB permeability of L-dopa for increasing DA level in PD treatment.Besides,the co-administration might contribute to ?-syn and inactive MEF2D degradation and eventually protect dopaminergic neurons from the damage induced by 6-OHDA via chaperone-mediated autophagy activation,HSP70 upregulation and macroautophagy inhibition in HSP70/MAPK/MEF2D/Beclin-1 pathway in PD rats. |
PDF Full Text Request