| Parkinson' s disease(PD)is a common neurodegenerative disease.At present,the etiology of PD is unknown,but no effective drug treatment,symptoms mainly controlled by levodopa and related preparations,but less effective,symptom fluctuation,movement disorders and other side effects after several years.Therefore,it is a most important task to research the effective drugs for the treatment of PD.Chinese traditional medicine for the treatment of PD has an outstanding advantage,which plays an important role in the occurrence and development of xingnaokaiqiao drug prevention and treatment of PD.Acorus tatarinowii is a representative drug of xingnaokaiqiao drug,has a prominent role in the treatment of nervous system diseases.It can improve neurological function,reduce brain edema and nerve damage to the central nervous system and brain cell ultrastructure have definite protective effect.Acorus tatarinowii volatile oil is the main active ingredient,and beta-asarone is the main component of Acorus tatarinowii volatile oil.Previous studies have found that beta-asarone can improve the behavior of 6-hydroxydopamine(6-OHDA)induced rats;increased dopamine(DA)in the striatum;reduce the toxicity of alpha-synuclein(alpha-syn)and increase tyrosine hydroxylase(TH)expression in the brain.Therefore,beta-asarone has an effective therapeutic effect on 6-OHDA induced rats.At present,madopar is the first-line drug for the clinical treatment of PD,whether the beta-asarone combined with small dose of madopar can improve the behavior of 6-OHDA induced rats,increasing DA and promote the synthesis of enzymes related to DA content?This can reduce the madopar dosage and application time of madopar.In addition,the endoplasmic reticulum stress and autophagy are closely related to the PD,it is worth studying that beta-asarone plays a protective role through endoplasmic reticulum stress and autophagy.The experiment was mainly composed of five parts,part I:To observe the behavior,neurotransmitters,DA related enzymes,liver and kidney function,and histopathology on 6-OHDA induced rats treated by beta-asarone combined with madopar.To investigate the efficacy of combination therapy with madopar can achieve the single level,so as to reduce the dosage of madopar,the purpose is reducing toxic and extending the efficiency of madopar.Part II:To observe the dynamic changes of alpha-syn,alpha-syn mRNA,beta-synuclein(beta-syn)mRNA,GRP78 mRNA and Beclin-1 mRNA on 6-OHDA induced rats in the midbrain,striatum and hypothalamus.To analyze the correlation between alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA,GRP78 mRNA and Beclin-1 mRNA,alpha-syn mRNA and alpha-syn protein levels.Park III:To observe the effect of beta-asarone on endoplasmic reticulum stress and IRE1/XBP1 pathway.Park IV:To observe the relationship between endoplasmic reticulum stress and autophagy on 6-OHDA induced rats.Park V:To observe beta-asarone modulates the PERK/CHOP/Bcl-2/Beclin-1 pathway on 6-OHDA induced rats.Part 11.1 ObjectivePrevious studies found that beta-asarone can improve the behavior of 6-OHDA induced rats;increase the levels of DA,homovanillic acid(HVA)and 5-hydroxyindoleacetic acid(5-HIAA);reduce the level of alpha-syn,and increase the expression of TH in the brain.In addition,beta-asarone can promote levodopa(L-DOPA)into the brain,increase the level of DA in the brain.To observe beta-asarone combined with madopar on 6-OHDA induced rats in this study.1.2 MethodsSeventy Sprague Dawley rats(both sexes each half),220-250 g,were taken from Guangzhou University of Chinese Medicine.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were placed in the flat skull position on a cotton bed on a stereotaxic frame(Stoelting,USA)with incisor bar fixed at 4.5 mm below the interaural line.Severe unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?l 6-OHDA(4 ?g/?l,free based dissolved in a solution of 0.2 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.The model rats were injected intraperitoneally with 0.5 mg/kg apomorphine in thirty days after modeling.The successful PD model rotated to the healthy side more than 7 cycles per minute.The successful model rats were randomly divided into 6 groups,each group of 10 rats.The model group respectively(1 ml/100 g,distilled water)and madopar group(75 mg/kg,madopar),beta-asarone group(15 mg/kg,beta-asarone),beta-asarone+1/4 madopar group(15 mg/kg,beta-asarone;18.75 mg/kg,madopar),beta-asarone+1/2 madopar group(15 mg/kg,beta-asarone;37.5 mg/kg,madopar),and beta-asarone+madopar group(15 mg/kg,beta-asarone;75 mg/kg,madopar).Intragastric administration was administered 2 times a day for 30 days.In the administration of 28 days of open field test,rotarod and gait training experiment,the behavior detect in 29 and 30 days.In 31 days,rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were taken out of the blood,the livers and kidneys were taken from the abdominal cavity,and the striatum and midbrain were taken in the brain.High performance liquid chromatography(HPLC)test L-DOPA,DA,DOPAC,5-HT,5-HIAA and HVA in the striatum;DA in serum;enzyme-linked immunosorbent assay(ELISA)test DDC in serum and brain;flow cytometry test TH;ELISA test the index of liver and kidney function;HE test liver and kidney tissue for histological examination.1.3 Results1.3.1 The behavioral effects of beta-asarone combined with madopar on 6-OHDA induced ratsCompared with the sham operated group,the number of moving squares,stands and the residence time of the rods were decreased(P<0.05),while the central residence time and the right forelimb start time increased(P<0.05).Compared with the model group,madopar group,beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group significantly increased in the number of moving squares(p<0.05);madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group significantly increased the number of standing(P<0.05);madopar group,beta-asarone group,beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group significantly increased stay rod time and stopping time,but the right forelimb start time was significantly reduced(P<0.05).Compared with madopar group,there was no significant difference in the number of moving squares in beta-asarone+1/2 madopar group and beta-asarone+madopar group(P>0.05);the number of standing,rotating rod retention time and right forelimb start time had no significantly difference in beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P>0.05);the central lattice retention time had no significantly difference in beta-asarone+madopar group(P>0.05).1.3.2 Biochemical analysisCompared with the sham operation group,the alanine amino transferase(ALT),aspartateaminotransferase(AST),creatinine(CR),blood urea nitrogen(BUN)and total bilirubin(TBIL)have no significantly difference in model group(P>0.05).Compared with the model group,the ALT and BUN significantly increased in madopar group,beta-asarone+1/2 madopar group and beta-asarone+mdopar group(P<0.05);the CR significantly increased in madopar group,beta-asarone+1/4 madopar group and beta-asarone+1/2 madopar group(P<0.05).1.3.3 Liver and kidney histopathologyThe liver tissue structure,liver cells arranged in neat,clear boundary,morphology,no fat vacuoles,portalarea no inflammatory cell infiltration in the sham operation group,model group and beta-asarone group.The structure of renal cortex and medulla is clear.No hyperplasia or atrophy of glomerular capillary loops,no necrosis,no glomerular capillary hyperemia,no thickening of wall.There was no inflammatory cell infiltration in renal interstitium in the sham operation group,model group and beta-asarone group.Liver tissue portal more inflammatory cell infiltration.Renal interstitial inflammatory cell infiltration in madopar group.The liver structure,liver cells arranged in neat,clear boundary,morphology,no fat vacuoles,portalarea showed different degrees of inflammatory cell infiltration in the combination group,beta-asarone+1/4 madopar group rats periportal inflammatory cells at least.Renal interstitial focal necrosis and inflammatory cell infiltration in beta-asarone+madopar group;renal interstitial inflammatory cells gradually decreased in beta-asarone+1/2 madopar group;renal interstitial inflammatory cells at least in beta-asarone+1/4 madopar group.1.3.4 The levels of neurotransmitters in the striatumCompared with the sham operation group rats,DA,DOPAC and 5-HT were significantly reduced in the model group(P<0.05).Compared with the model group,L-DOPA was significantly increased in the madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P<0.05);DA and DOPAC was significantly increased in the madopar group,beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P<0.05);the 5-HT was significantly increased in the beta-asarone+1/4 madopar group and beta-asarone+1/2 madopar group(P<0.05).Compared with madopar group,L-DOPA was not significantly difference in beta-asarone+ 1/2 madopar group(P>0.05);DA was not signifycantly difference in beta-asarone,beta-asarone+1/4 madopar group and beta-asarone+ madopar group(P>0.05);DA was significantly increased in the beta-asarone+1/2 madopar group(P<0.05);DOPAC was not signifycantly difference in beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(/P>0.05);5-HT was not signifycantly difference in beta-asarone,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P>0.05);5-HT was significantly increased in the beta-asarone+1/4 madopar group(P<0.05);5-HIAA and HVA was not signifycantly difference in beta-asarone,beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P>0.05).1.3.5 The DA in serumCompared with the sham operation group,the DA had no significant difference in model group(P>0.05).Compared with the model group,DA was significantly increased in the madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P<0.05).Compared with the madopar group rats,DA had no significant difference in beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P>0.05).1.3.6 The changes of the related enzymesCompared with the sham operation group,the expression rate of TH was significantly decreased in model group(P<0.05).Compared with the model group,the expression rate of TH was significantly increased in madopar group and beta-asarone + 1/2 madopar group(P<0.05).There was no significant change of DDC in each group(P>0.05).In the serum,compared with sham operation group,DDC showed no significant change in the model group(P>0.05).Compared with the model group rats,DDC was significantly decreased in madopar group,beta-asarone group,beta-asarone+1/4 madopar group,beta-asarone+1/2 madopar group and beta-asarone+madopar group(P<0.05).1.4 ConclusionsIn conclusion,the combined therapy improves the behavior of 6-OHDA induced rats,there was not significant difference between beta-asarone+1/2 madopar group and madopar group.The combined therapy reduces the damage of liver and kidney function.The combined therapy increases L-DOPA,DA,DOPAC and 5-HT in the striatum;DA increase in serum.The combination therapy had little effect on TH in midbrain.The combination therapy inhibits DDC in serum,there was little effect on DDC in midbrain.Part 22.1 ObjectiveThe preliminary study found that alpha-syn is the main component of Lewy body,and lewy body caused the death of dopaminergic neurons.Therefore,alpha-syn is the pathological basis of PD.Other studies have shown that beta-syn can reduce the alpha-syn damage to dopaminergic neurons,the endoplasmic reticulum stress and autophagy in the pathological process of PD.This study observed the dynamic changes of alpha-syn mRNA,beta-syn mRNA,endoplasmic reticulum stress protein GRP78 mRNA and autophagy protein Beclin-1 mRNA in the midbrain,striatum and hypothalamus of 6-OHDA induced rats,the dynamic changes of alpha-syn in the midbrain and striatum of 6-OHDA induced rats.To analyze the correlation between alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA,GRP78 mRNA and Beclin-1 mRNA,alpha-syn mRNA and alpha-syn protein levels.2.2 MethodsSixty Sprague Dawley rats(both sexes each half),220-250 g,were taken from Guangzhou University of Chinese Medicine.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were placed in the flat skull position on a cotton bed on a stereotaxic frame(Stoelting,USA)with incisor bar fixed at 4.5 mm below the interaural line.Severe unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?1 6-OHDA(4 ?g/?l,free based dissolved in a solution of 0.2 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.The rats were divided into sham operation group,3 h group,12 h group,24 h group,72 h group and 144 h group with 10 rats in each group.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.),midbrain,striatum and hypothalamus were harvested.RT-PCR detect the alpha-syn mRNA,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA in midbrain,striatum and hypothalamus;and the Pearson correlation between alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA,Beclin-1 mRNA and GRP78 mRNA,alpha-syn mRNA and alpha-syn protein levels.ELISA test alpha-syn in midbrain and striatum.2.3 Results2.3.1 Dynamic changes of alpha-syn mRNA,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA in mesencephalons,striatum and hypothalamusThe levels of alpha-syn mRNA,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA were low in the sham group,but rose significantly after 6-OHDA injected(P<0.05).In mesencephalons,alpha-syn mRNA peaked at 3 h,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA peaked at 12 h.After the peak,they declined slowly until 144 h.In striatum,alpha-syn mRNA peaked at 3 h,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA peaked at 12 h.After the peak,they declined slowly until 72 h,but increased again at 144 h.In hypothalamus,all of them peaked at 3 h.After the peak,they declined slowly until 24 h,but increased again at 72 h.2.3.2 Dynamic expressions of alpha-syn in mesencephalons and striatumThe expression of alpha-syn was low in the sham group,but rose significantly after 6-OHDA injected(P<0.05).In mesencephalons,alpha-syn peaked at 3 h.After the peak,it declined slowly until 144 h.In striatum,alpha-syn peaked at 3 h.After the peak,it declined slowly until 72 h.But it increased again at 144 h2.3.3 Correlation between alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA,GRP78 mRNA and Beclin-1 mRNAIn mesencephalon,Pearson analysis showed that alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA were significant positive correlations between 0 and 3 h,between 12 h and 144 h,aSig.(2-tailed)P<0.05.In contrast,their relationships were significant negative correlations between 3 h and 12 h,aSig.(2-tailed)P<0.05.Pearson analysis showed that correlations between GRP78 mRNA and Beclin-1 mRNA expressions were significantly positive correlations between 0 and 3 h,between 12 h and 144 h,aSig.(2-tailed)P<0.05.In striatum,Pearson analysis showed that alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,alpha-syn mRNA and Beclin-1 mRNA were significant positive correlations between 0 and 3 h,between 12 h and 144 h,aSig.(2-tailed)P<0.05.In contrast,their relationships were significant negative correlations between 3 h and 12 h,aSig.(2-tailed)P<0.05.Pearson analysis showed that correlations between GRP78 mRNA and Beclin-1 mRNA were significantly positive between 0 and 144 h,aSig.(2-tailed)P<0.05.In hypothalamus,Pearson analysis showed that alpha-syn mRNA and beta-syn mRNA,alpha-syn mRNA and GRP78 mRNA,GRP78 mRNA and Beclin-1 mRNA were significant positive correlations between 0 and 144 h,aSig.(2-tailed)P<0.05.alpha-Syn mRNA and Beclin-1 mRNA were significant positive correlations between 3 h and 144 h,aSig.(2-tailed)P<0.05.2.3.4 Correlation between alpha-syn mRNA and alpha-syn protein levelsIn mesencephalon and striatum,Pearson analysis showed that alpha-syn mRNA and alpha-syn protein was significant positive correlations between 0 and 144 h,aSig.(2-tailed)P<0.05.2.4 ConclusionsIn conclusion,the levels of alpha-syn mRNA,beta-syn mRNA,GRP78 mRNA and Beclin-1 mRNA were dynamically expressed in different brain parts after 6-OHDA injected.The alpha-syn increased in the early stage of PD,which induced the expression of beta-syn,GRP78 and Beclin-1.Subsequently,the expression of beta-syn,GRP78 and Beclin-1 increased,and the expression of alpha-syn was decreased.Part 33.1 ObjectiveThe preliminary study found that endoplasmic reticulum stress has close relationship with PD,and involved in the pathological process of PD,there was positive correlation between alpha-syn mRNA and GRP78 mRNA.In this experiment,to observe beta-asarone inhibits IRE1/XBP1 endoplasmic reticulum stress pathway in 6-OHDA-induced rats.3.2 MethodsSixty Sprague Dawley rats(both sexes each half),220-250 g,were taken from Guangzhou University of Chinese Medicine.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were placed in the flat skull position on a cotton bed on a stereotaxic frame(Stoelting,USA)with incisor bar fixed at 4.5 mm below the interaural line.Severe unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?l 6-OHDA(4 ?g/?l,free based dissolved in a solution of 0.2 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.The model rats were injected intraperitoneally with 0.5 mg/kg apomorphine in thirty days after modeling.The successful PD model rotated to the healthy side more than 7 cycles per minute.The successful model rats were randomly divided into 5 groups,each group of 10 rats.Sham-operated group was given normal saline;model group(6-OHDA)was given 6-OHDA lesion,thirty days after modeling,followed by normal saline(intragastric administration);beta-asarone group(6-OHDA+beta-asarone)was given by 6-OHDA lesion,thirty days after modeling,followed by 15 mg/kg of beta-asarone intragastricly per day;4-phenylbutyric acids(4-PBA)group(6-OHDA+4-PBA)was given by 6-OHDA lesion,thirty days after modeling,followed by 100 mg/kg of 4-PBA---endoplasmic reticulum stress inhibitor,intraperitoneal injection per day;tunicamycin(TM)group(6-OHDA+TM)was given by 6-OHDA lesion,thirty days after modeling,followed by 0.5 mg/kg of TM---endoplasmic reticulum stress inducer,intraperitoneal injection per week;IRE1 inhibitor group(6-OHDA+STF-083110)was given by 6-OHDA lesion,thirty days after modeling,followed by 15 mg/kg of STF-083110---IRE1 inhibitor,intraperitoneal injection per day.All groups were continuously administered for 4 weeks.The striatum and mesocerebrum were harvested and stored at-80? for RT-PCR and western blot analysis.3.3 Results3.3.1 The levels of GRP78 mRNA and CHOP mRNA in striatum after different treatmentsA significant increase of GRP78 mRNA and CHOP mRNA levels were found in model group compared with that in sham-operated group(P<0.05).On the contrary,GRP78 mRNA and CHOP mRNA levels showed a significant decrease in beta-asarone group and 4-PBA group compared with that in model group(P<0.05).The levels of GRP78 mRNA and CHOP mRNA have no significant difference in beta-asarone group and 4-PBA group(P>0.05).3.3.2 The expressions of p-IREl and XBP1 in mesocerebrum after different treatmentsA significant increase of p-IRE1 and XBP1 expression were found in 6-OHDA model group compared with that in sham-operated group(P<0.05).On the contrary,p-IRE1 and XBP1 expression showed a significant decrease in beta-asarone group and IRE1 inhibitor group compared with that in 6-OHDA model group(P<0.05).The expressions of p-IRE1 and XBP1 have no significant difference in beta-asarone group and IRE1 inhibitor group(P>0.05).3.3.3 The levels of IRE1 mRNA and XBP1 mRNA in mesocerebrum after different treatmentsA significant increase of IRE1 mRNA and XBP1 mRNA levels were found in model group compared with that in sham-operated group(P<0.05).On the contrary,IRE1 mRNA and XBP1 mRNA levels showed a significant decrease in beta-asarone group and IRE1 inhibitor group compared with that in model group(P<0.05).The levels of IRE1 mRNA and XBP1 mRNA have no significant difference in beta-asarone group and IRE1 inhibitor group(P>0.05).3.4 ConclusionsBeta-asarone has neural protective effects on 6-OHDA induced pakinsonian rats through inhibiting IRE1/XBP1 endoplasmic reticulum stress pathway,and it can be used as a potential drug for the treatment of PD.Part 44.1 ObjectiveStudies have shown that endoplasmic reticulum stress and autophagy are closely related to PD,and are involved in the pathological process of PD.Other studies have shown that endoplasmic reticulum stress induces autophagy.This study was conducted to investigate the relationship between endoplasmic reticulum stress and autophagy in 6-OHDA induced rats.4.2 MethodsSixty Sprague Dawley rats(both sexes each half),220-250 g,were taken from Guangzhou University of Chinese Medicine.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were placed in the flat skull position on a cotton bed on a stereotaxic frame(Stoelting,USA)with incisor bar fixed at 4.5 mm below the interaural line.Severe unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?l 6-OHDA(4 ?g/?l,free based dissolved in a solution of 0.2 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.The model rats were injected intraperitoneally with 0.5 mg/kg apomorphine in thirty days after modeling.The successful PD model rotated to the healthy side more than 7 cycles per minute.The successful model rats were randomly divided into 5 groups,each group of 10 rats.Sham-operated group was given 0.2 mg/ml L-ascorbic acid in normal saline,followed by normal saline,intraperitoneal injection per day;model group(6-OHDA)was given 6-OHDA lesion,followed by normal saline,intraperitoneal injection per day;3-methyladenine(3-MA)group(6-OHDA+3-MA)was given by 6-OHDA lesion,followed by 3 mg/kg of 3-MA---autophagy inhibitor,intraperitoneal injection per day;rapamycin group(6-OHDA+rapamycin)was given by 6-OHDA lesion,followed by 1 mg/kg of rapamycin---autophagy inducer,intraperitoneal injection per day;4-phenylbutyric acids(4-PBA)group(6-OHDA+4-PBA)was given by 6-OHDA lesion,followed by 100 mg/kg of 4-PBA---endoplasmic reticulum stress inhibitor,intraperitoneal injection per day;tunicamycin(TM)group(6-OHDA+TM)was given by 6-OHDA lesion,followed by 0.5 mg/kg of TM---endoplasmic reticulum stress inducer,intraperitoneal injection per week.Thirty days after modeling,all groups were continuously administered for 4 weeks.4.3 Results4.3.1 The levels of GRP78 mRNA and Beclin-1 mRNAA significant increase of GRP78 mRNA and Beclin-1 mRNA levels were found in model group compared with that in sham-operated group(P<0.05).The Beclin-1 mRNA levels showed a significant decrease in 3-MA group compared with that in model group(P<0.05),and the Beclin-1 mRNA levels showed a significant increase in rapamycin group compared with that in model group(P<0.05),but the GRP78 mRNA levels in 3-MA and rapamycin group have no significant difference compared with that in model group(P>0.05).The GRP78 mRNA and Beclin-1 mRNA levels showed a significant decrease in 4-PBA group compared with that in model group(P<0.05).On the contrary,the GRP78 mRNA and Beclin-1 mRNA levels showed a significant increase in TM group compared with that in model group(P<0.05).4.3.2 The expressions of GRP78 and Beclin-1A significant increase of GRP78 and Beclin-1 were found in model group compared with that in sham-operated group(P<0.05).The Beclin-1 showed a significant decrease in 3-MA group compared with that in model group(P<0.05),and the Beclin-1 showed a significant increase in rapamycin group compared with that in model group(P<0.05),but the GRP78 have no significant difference in 3-MA and rapamycin group compared with that in model group(P>0.05).The expressions of GRP78 and Beclin-1 showed a significant decrease in 4-PBA group compared with that in model group(P<0.05).On the contrary,the expressions of GRP78 and Beclin-1 showed a significant increase in TM group compared with that in model group(P<0.05).4.4 ConclusionsThe relationship between endoplasmic reticulum stress and autophagy is very important in PD.In this study,we report that endoplasmic reticulum stress may induce autophagy in 6-OHDA-induced rats.Part 55.1 ObjectiveOur research group found that beta-asarone plays a neuroprotective role through the JNK/Bcl-2/Beclin-1 autophagy pathway.Former experiments showed that endoplasmic reticulum stress may induce autophagy.In this experiment,to observe whether beta-asarone may regulate the endoplasmic reticulum stress-autophagy via PERK/CHOP/Bcl-2/Beclin-1 pathway in 6-OHDA-induced rats.5.2 MethodsForty Sprague Dawley rats(both sexes each half),220-250 g,were taken from Guangzhou University of Chinese Medicine.Rats were anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg,i.p.)and were placed in the flat skull position on a cotton bed on a stereotaxic frame(Stoelting,USA)with incisor bar fixed at 4.5 mm below the interaural line.Severe unilateral lesion of the nigrostriatal pathway was obtained by micro-injection(SGE,Australia)of 6 ?l 6-OHDA(4 ?g/?l,free based dissolved in a solution of 0.2 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.The model rats were injected intraperitoneally with 0.5 mg/kg apomorphine in thirty days after modeling.The successful PD model rotated to the healthy side more than 7 cycles per minute.The successful model rats were randomly divided into 3 groups,each group of 10 rats.Sham-operated group was given normal saline;model group(6-OHDA)was given 6-OHDA lesion,thirty days after modeling,followed by normal saline(intragastric administration);beta-asarone group(6-OHDA+beta-asarone)was given by 6-OHDA lesion,thirty days after modeling,followed by 15 mg/kg of ?-asarone intragastricly per day;PERK inhibitor group(6-OHDA+ISRIB)was given by 6-OHDA lesion,thirty days after modeling,followed by 0.125 mg/kg of ISRIB intraperitoneal injection per day.All groups were continuously administered for 30 days.The mesencephalons and striatums were harvested for flow cytometry,RT-PCR and western blot analysis.5.3 Results5.3.1 The expressions of GRP78,p-PERK and CHOP in striatumsA significant increase of GRP78,p-PERK and CHOP expressions were found in 6-OHDA model group compared with that in sham-operated group(P<0.05).On the contrary,GRP78,p-PERK and CHOP expressions showed a significant decrease in beta-asarone group and PERK inhibitor group compared with that in 6-OHDA model group(P<.05).The expressions of GRP78,p-PERK and CHOP have no significant difference in beta-asarone group and PERK inhibitor group(P>0.05).5.3.2 The levels of GRP78 mRNA,PERK mRNA and CHOP mRNA in mesencephalonsA significant increase of GRP78 mRNA,PERK mRNA and CHOP mRNA levels were found in 6-OHDA model group compared with that in sham-operated group(P<0.05).On the contrary,GRP78 mRNA,PERK mRNA and CHOP mRNA levels showed a significant decrease in beta-asarone group and PERK inhibitor group compared with that in 6-OHDA model group(P<0.05).The levels of GRP78 mRNA,PERK mRNA and CHOP mRNA have no significant difference in beta-asarone group and PERK inhibitor group(P>0.05).5.3.3 The expressions of Bcl-2 and Beclin-1 in striatumsA significant increase of Beclin-1 expression but a decrease of Bcl-2 were found in 6-OHDA model group compared with that in sham-operated group(P<0.05).On the contrary,Beclin-1 expression showed a significant decrease in beta-asarone group and PERK inhibitor group compared with that in 6-OHDA model group(P<0.05).Whereas the beta-asarone group and PERK inhibitor group rats showed a significant increase of Bcl-2 expression compared with that in 6-OHDA model group(P<0.05).The expressions of Bcl-2 and Beclin-1 have no significant difference in beta-asarone group and PERK inhibitor group(P>0.05).5.3.4 The expressions of Bcl-2 and Beclin-1 in mesencephalonsA significant increase of Beclin-1 expression but a decrease of Bcl-2 were found in 6-OHDA model group compared with that in sham-operated group(P<0.05).On the contrary,Beclin-1 expression showed a significant decrease in beta-asarone group and PERK inhibitor group compared with that in 6-OHDA model group(P<0.05).Whereas the beta-asarone group and PERK inhibitor group rats showed a significant increase of Bcl-2 expression compared with that in 6-OHDA model group(P<0.05).The expressions of Bcl-2 and Beclin-1 have no significant difference in beta-asarone group and PERK inhibitor group(P>0.05).5.4 ConclusionBeta-asarone might inhibit PERK and regulate the endoplasmic reticulum stress-autophagy via PERK/CHOP/Bcl-2/Beclin-1 pathway.Final ConclusionsBeta-asarone combined with small dose of madopar can improve the behavior of 6-OHDA induced rats,reduce the damage of liver and kidney function,increase the L-DOPA,DA.DOPAC and 5-HT in the striatum,increase DA in serum,inhibit DDC in serum.In the early of oxidative stress,alpha-syn increase,beta-syn,GRP78 and Beclin-1 expression may be induced by alpha-syn.Subsequently,the expression of beta-syn,GRP78 and Beclin-1 increased,and the expression of alpha-syn was decreased.Endoplasmic reticulum stress may induce autophagy in 6-OHDA induced rats.Beta-asarone may inhibit IRE1/XBP1 endoplasmic reticulum stress pathway and may inhibit PERK/CHOP/Bcl-2/Beclin-1 of endoplasmic reticulum stress autophagy pathway. |
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