nurr1 also known as nr4a2 , belongs to the nuclear receptor superfamily of transcription factors , is highly expressed in midbrain dopaminergic ( da ) neurons , and plays an essential role in the development , survival and function maintenance of midbrain da neurons . nurr1 is a potential target for parkinson 's disease ( pd ) , which is the second common neurodegenerative disease and both genetic and environmental neurotoxin are important causes . nurr1 can activate the transcription of tyrosine hydroxylase and enhance the expression of da transporter . it was reported that homozygous nurr1 knockout mice failed to develop da neurons , and heterozygotes reduced their brain da and developed age - dependent locomotor deficits including impaired horizontal and vertical movement , difficultly performance on rotarod test compared to age - matched wild - type mice . reduction of nurr1 in the adult brain may increase the vulnerability of da neurons to stress and participate in the pathogenesis of pd . several polymorphisms of nurr1 gene including 291tdel and 245t-->g have been reported to result in a marked decrease of nurr1 mrna levels in transfected cell lines and lymphocytes of affected individuals . furthermore , le et al . reported a decreased nurr1 gene expression in peripheral blood mononuclear cells ( pbmcs ) of pd patients , which anticipated nurr1 as a potential biomarker for pd diagnosis . but to date , little has been done to investigate the association among nurr1 expression in pbmc , disease progression of pd , and da agents . this study was to measure the nurr1 mrna level in pbmc and evaluate the effect of nurr1 expression by da agents in vivo and in vitro . the 362 pd patients at department of neurology , the first affiliated hospital of sun yet - sen university between january 2012 and august 2014 were recruited . the diagnosis was made in accordance with the united kingdom pd society brain bank criteria . besides recent - onset pd patients without any anti - pd treatments ( de novo ) , each patient reported to have taken anti - pd medicines at least 2 years and all patients were assessed according to the hoehn and yahr ( h and y ) scale . meantime , 193 healthy controls ( hcs ) were enrolled either from physical examination center of sun yat - sen university or the spouses of the patients . the study was approved by the ethics committee of sun yat - sen university . peripheral blood samples ( 5 ml ) of pd patients and hcs pbmc from venous blood of pd patients and hcs were prepared by ficoll - hypaque density gradient centrifugation . after centrifugation at 2200 r / min for 20 min , buffy coats were collected and washed three times with phosphate - buffered saline ( pbs ) , then centrifuged at 1800 r / min for 10 min to collect pbmc cells . then pbmc were lysed by adding 1 ml trizol reagent ( invitrogen , carlsbad , ca , usa ) and stored at 80c until used . in order to further explore the pattern of nurr1 expression , an in vitro analysis on cultured human pbmc was conducted . pbmc from venous blood of 10 hcs was prepared by ficoll - hypaque density gradient centrifugation through centrifuging at 2200 r / min for 20 min . the buffy coats were collected and washed twice with pbs , then washed once with complete rpmi 1640 medium ( gibco , german ) containing 2 mmol / l l - glutamine , 100 u / ml penicillin , 100 g / ml streptomycin , and 10% fetal bovine serum and suspended at a concentration of 2  10 cells / ml with complete rpmi 1640 medium . pbmc was delivered in 3.5 cm diameter dishes with 2  10 cells separately with complete rpmi 1640 medium . after 12 h cultured , the cells were treated with 10 mol / l pramipexole ( sigma , st . louis , mo , usa ) for 2 , 4 , 8 , 12 and 24 h and cultured at 37c under the humidified 5% co2 atmosphere . selected time , the pbmc cells were collected for rna extraction . then total rna was isolated using trizol ( invitrogen ) according to the manufacturer 's protocol . the 1 g of total rna was reverse transcribed into first - strand complementary dna ( cdna ) using a reverse transcription ( rt ) kit ( takara , japan ) according to the manufacturer 's protocol in a final volume of 20 l . for analysis , on nurr1 expression with quantitative real - time rt - polymerase chain reaction ( rt - pcr ) , human glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was used as internal control , and the sequences of primers as follows : nurr1 forward : 5-tccaacgaggggctgtgcg-3 ; nurr1 reverse : 5-cactgtgcgcttaaagaagc-3 ; and gapdh forward : 5-gaaggtgaagg tcggagtc-3 ; gapdh reverse : 5-gaagatggtgatgggatttc-3. the 2 l of the synthesized cdna were used in all real - time pcr together with the sybr green i master mix ( sybr premix ex taq , takara , japan ) on mj research opticon2 real - time thermocycler ( bio - rad , hercules , ca , usa ) . fluorescent reading from real - time pcr reaction was quantitatively analyzed by determining the difference of ct ( delta ct ) between ct of nurr1 and its internal gapdh , and the nurr1 gene expression was determined by the formation of 2 . in addition , for all the analyzed samples , only triplicates with a standard deviation of the ct < 0.20 were accepted . quantitative data ( age , h and y scale , the relative nurr1 mrna relative level ) were shown as mean  standard error ( se ) or median and interquartile range , depending on the distribution of the data . the chi - square test was used to analyze the differences of gender between pd and hcs or among the subgroups of pd . the nonparametric mann - whitney u - test and kruskal - wallis test were performed to evaluate the differences between pd and hc or among the subgroups of pd . the multivariate linear regression models were used to simultaneously evaluate the cross - sectional association of dependent variables ( the nurr1 mrna relative level ) and independent variables ( h and y scale , age , and the different drug treatments ) . after adjustment of h and y scale and age , the covariance analysis was utilized to test the differences of nurr1 mrna relative level among different drug treatments . one - way analysis of variance was used to analyze the difference of nurr1 mrna level among the different hours treated with pramipexole . the 362 pd patients at department of neurology , the first affiliated hospital of sun yet - sen university between january 2012 and august 2014 were recruited . the diagnosis was made in accordance with the united kingdom pd society brain bank criteria . besides recent - onset pd patients without any anti - pd treatments ( de novo ) , each patient reported to have taken anti - pd medicines at least 2 years and all patients were assessed according to the hoehn and yahr ( h and y ) scale . meantime , 193 healthy controls ( hcs ) were enrolled either from physical examination center of sun yat - sen university or the spouses of the patients . peripheral blood samples ( 5 ml ) of pd patients and hcs were drawn from antecubital venous and put into natrium citricum - containing tubes . pbmc from venous blood of pd patients and hcs were prepared by ficoll - hypaque density gradient centrifugation . after centrifugation at 2200 r / min for 20 min , buffy coats were collected and washed three times with phosphate - buffered saline ( pbs ) , then centrifuged at 1800 r / min for 10 min to collect pbmc cells . then pbmc were lysed by adding 1 ml trizol reagent ( invitrogen , carlsbad , ca , usa ) and stored at 80c until used . in order to further explore the pattern of nurr1 expression , an in vitro analysis on cultured human pbmc was conducted . pbmc from venous blood of 10 hcs was prepared by ficoll - hypaque density gradient centrifugation through centrifuging at 2200 r / min for 20 min . the buffy coats were collected and washed twice with pbs , then washed once with complete rpmi 1640 medium ( gibco , german ) containing 2 mmol / l l - glutamine , 100 u / ml penicillin , 100 g / ml streptomycin , and 10% fetal bovine serum and suspended at a concentration of 2  10 cells / ml with complete rpmi 1640 medium . pbmc was delivered in 3.5 cm diameter dishes with 2  10 cells separately with complete rpmi 1640 medium . after 12 h cultured , the cells were treated with 10 mol / l pramipexole ( sigma , st . louis , mo , usa ) for 2 , 4 , 8 , 12 and 24 h and cultured at 37c under the humidified 5% co2 atmosphere . then total rna was isolated using trizol ( invitrogen ) according to the manufacturer 's protocol . the 1 g of total rna was reverse transcribed into first - strand complementary dna ( cdna ) using a reverse transcription ( rt ) kit ( takara , japan ) according to the manufacturer 's protocol in a final volume of 20 l . for analysis , on nurr1 expression with quantitative real - time rt - polymerase chain reaction ( rt - pcr ) , human glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was used as internal control , and the sequences of primers as follows : nurr1 forward : 5-tccaacgaggggctgtgcg-3 ; nurr1 reverse : 5-cactgtgcgcttaaagaagc-3 ; and gapdh forward : 5-gaaggtgaagg tcggagtc-3 ; gapdh reverse : 5-gaagatggtgatgggatttc-3. the 2 l of the synthesized cdna were used in all real - time pcr together with the sybr green i master mix ( sybr premix ex taq , takara , japan ) on mj research opticon2 real - time thermocycler ( bio - rad , hercules , ca , usa ) . fluorescent reading from real - time pcr reaction was quantitatively analyzed by determining the difference of ct ( delta ct ) between ct of nurr1 and its internal gapdh , and the nurr1 gene expression was determined by the formation of 2 . in addition , for all the analyzed samples , only triplicates with a standard deviation of the ct < 0.20 were accepted . quantitative data ( age , h and y scale , the relative nurr1 mrna relative level ) were shown as mean  standard error ( se ) or median and interquartile range , depending on the distribution of the data . the chi - square test was used to analyze the differences of gender between pd and hcs or among the subgroups of pd . the nonparametric mann - whitney u - test and kruskal - wallis test were performed to evaluate the differences between pd and hc or among the subgroups of pd . the multivariate linear regression models were used to simultaneously evaluate the cross - sectional association of dependent variables ( the nurr1 mrna relative level ) and independent variables ( h and y scale , age , and the different drug treatments ) . after adjustment of h and y scale and age , the covariance analysis was utilized to test the differences of nurr1 mrna relative level among different drug treatments . one - way analysis of variance was used to analyze the difference of nurr1 mrna level among the different hours treated with pramipexole . the 362 pd patients ( 209 males , 153 females ) were enrolled in this study . among these 362 patients , 136 patients were recent - onset cases and did not receive any anti - pd treatment before blood drawing ( de novo ) ; 83 patients were administrated with l - dopa , 38 patients received d2/d3 agonists ( piribedil or pramipexole ) , 105 patients took both l - dopa and da agonists . these patients were divided into four subgroups according to the different drug treatments : de novo , l - dopa , da agonist , and l - dopa and da agonist . the demographic and clinical characteristics of pd patients and hcs are summarized in table 1 , showing no significant differences ( p = 0.683 ) in gender between pd patients and hcs and among the four subgroups of pd patients . the average age of pd patients in l - dopa and l - dopa and da agonist subgroups was older than that of de novo subgroup or hcs ( p < 0.05 ) . furthermore , the median scale of pd was 2.0 of h and y stage in all three subgroups ( l - dopa , da agonists and l - dopa and da agonist ) , which was significantly higher than de novo subgroup ( p < 0.05 ) . demographic and clinical characteristics of the subjects da : dopaminergic ; hc : healthy control ; pd : parkinson s disease ; de novo : pd without any anti - pd treatment ; da agonists : pd treated with da agonists ; l - dopa : pd treated with l - dopa ; l - dopa and da agonists : pd treated with l - dopa and da agonists . * p<0.05 compared with de novo ; p<0.05 compared with hc . generalized estimating equations in linear regression models were performed on the data from all subjects . h and y scale , age and different drug treatments of pd patients were identified to be independently associated with nurr1 mrna relative level . in both the stepwise model and inclusive model , the independent predictors of nurr1 mrna relative level were da agonists , l - dopa , and da agonists . the formulae for nurr1 mrna relative level were created based on the regression coefficients (  ) : in stepwise model , for da agonists ,  = 4.551 ( 95% confidence interval [ ci ] , 2.2006.903 ; p < 0.001 ) ; for l - dopa and da agonists ,  = 4.398 ( 95% ci , 2.8835.912 ; p < 0.001 ) ; and in inclusive model , for age ,  = 0.024 ( 95% ci , 0.0690.022 ; p = 0.311 ) ; for h and y scale ,  = 0.120 ( 95% ci , 1.0920.852 ; p = 0.809 ) ; for de novo ,  = 0.633 ( 95% ci , 2.7391.473 ; p = 0.555 ) ; for l - dopa ,  = 0.672 ( 95% ci , 2.0583.402 ; p = 0.629 ) ; for da agonists ,  = 4.657 ( 95% ci , 1.5527.762 ; p = 0.003 ) ; for l - dopa and da agonists ,  = 4.561 ( 95% ci , 1.9737.147 ; p = 0.001 ) . the nurr1 mrna relative level was made using the following formulas : nurr1 mrna level = 2.973 + 4.551  da agonists + 4.398  l - dopa and da agonists , or nurr1 mrna level = 4.545 + 4.657  da agonists + 4.561  l - dopa and da agonists . according to different drug treatments , the pd patients were divided into 4 subgroups . as shown in figure 1 , the relative levels of nurr1 mrna in de novo subgroup and hcs were 2.38  0.42 versus 3.18  0.42 , respectively ( p = 0.307 ) , a slight decrease but no statistical significance in de novo subgroup without any da agonists , l - dopa or other anti - pd drugs . the relative level of nurr1 mrna in l - dopa pd group was 3.37  0.57 versus 3.18  0.42 in hc ( p = 0.841 ) , which showed no statistical significance . however , the relative nurr1 mrna level in da agonist subgroup was 7.52  1.82 , significant higher than those both in de novo group ( p < 0.001 ) and hc ( p = 0.001 ) . the nurr1 mrna level in da agonist and l - dopa subgroup was up to 7.37  1.07 , which was significantly increased versus de novo subgroup or l - dopa subgroup ( p < 0.001 ) . there were no significant differences of nurr1 mrna level between da agonist and da agonist and l - dopa subgroups . scatter plots of nurr1 mrna relative levels in peripheral blood mononuclear cells of subgroups of pd patients . fluorescent reading from real - time polymerase chain reaction was quantitatively analyzed by determining the difference of ct ( delta ct ) between ct of nurr1 and internal control . hc : healthy control ; pd : parkinson 's disease ; de novo : pd without any anti - pd treatment ; da agonists : pd treated with da agonists ; l - dopa : pd treated with l - dopa ; l - dopa and da agonists : pd treated with l - dopa and da agonists ; da : dopaminergic . in 136 recent - onset cases , 25 were treated with l - dopa and 19 were treated with da agonists . the nurr1 mrna level in 25 patients was 7.87  1.70 before l - dopa treatment , and 7.59  1.59 after l - dopa taken , but no significant difference ( p = 0.905 ) . the nurr1 mrna levels in 19 patients before and after da agonist treatment were 5.92  1.42 and 8.47  2.52 , respectively , indicating a slight but nonsignificant increase after da agonist treatment ( p = 0.066 ) . scatter plots of nurr1 mrna relative levels in peripheral blood mononuclear cells before and after treatment of l - dopa ( a ) ( n = 25 ) or da agonist ( b ) ( n = 19 ) in de novo subgroup . there were no obvious changes before and after l - dopa treatment neither for the whole group nor for each individual , while there were three individuals showed obvious increase of nurr1 mrna levels though there was no significance between before and after da agonist treatment as a whole group . da : dopaminergic . to further explore whether da agonists have effect on nurr1 expression , we measured the nurr1 mrna level in pbmc from 10 healthy volunteers with 10 mol / l pramipexole [ figure 3 ] . compared with control , the nurr1 mrna relative levels in pbmc by da agonist were significantly increased to 199.61  13.17% , 171.75  11.33% , 173.16  11.42% in 2 , 4 and 8 h , respectively ( p < 0.001 ) . in vitro experiment , the nurr1 mrna levels of pbmc treated by da agonist for 12 h and 24 h were 95.30  6.26% and 104.60  4.32% , respectively , and there were no significant differences compared with control ( p > 0.05 ) , which was in accord with the half - life of 812 h. nurr1 mrna relative levels in vitro measured by real - time polymerase chain reaction treated with pramipexole . all experiments were performed at least three independent times ( * p < 0.001 ) . the 362 pd patients ( 209 males , 153 females ) were enrolled in this study . among these 362 patients , 136 patients were recent - onset cases and did not receive any anti - pd treatment before blood drawing ( de novo ) ; 83 patients were administrated with l - dopa , 38 patients received d2/d3 agonists ( piribedil or pramipexole ) , 105 patients took both l - dopa and da agonists . these patients were divided into four subgroups according to the different drug treatments : de novo , l - dopa , da agonist , and l - dopa and da agonist . the demographic and clinical characteristics of pd patients and hcs are summarized in table 1 , showing no significant differences ( p = 0.683 ) in gender between pd patients and hcs and among the four subgroups of pd patients . the average age of pd patients in l - dopa and l - dopa and da agonist subgroups was older than that of de novo subgroup or hcs ( p < 0.05 ) . furthermore , the median scale of pd was 2.0 of h and y stage in all three subgroups ( l - dopa , da agonists and l - dopa and da agonist ) , which was significantly higher than de novo subgroup ( p < 0.05 ) . demographic and clinical characteristics of the subjects da : dopaminergic ; hc : healthy control ; pd : parkinson s disease ; de novo : pd without any anti - pd treatment ; da agonists : pd treated with da agonists ; l - dopa : pd treated with l - dopa ; l - dopa and da agonists : pd treated with l - dopa and da agonists . generalized estimating equations in linear regression models were performed on the data from all subjects . h and y scale , age and different drug treatments of pd patients were identified to be independently associated with nurr1 mrna relative level . in both the stepwise model and inclusive model , the independent predictors of nurr1 mrna relative level were da agonists , l - dopa , and da agonists . the formulae for nurr1 mrna relative level were created based on the regression coefficients (  ) : in stepwise model , for da agonists ,  = 4.551 ( 95% confidence interval [ ci ] , 2.2006.903 ; p < 0.001 ) ; for l - dopa and da agonists ,  = 4.398 ( 95% ci , 2.8835.912 ; p < 0.001 ) ; and in inclusive model , for age ,  = 0.024 ( 95% ci , 0.0690.022 ; p = 0.311 ) ; for h and y scale ,  = 0.120 ( 95% ci , 1.0920.852 ; p = 0.809 ) ; for de novo ,  = 0.633 ( 95% ci , 2.7391.473 ; p = 0.555 ) ; for l - dopa ,  = 0.672 ( 95% ci , 2.0583.402 ; p = 0.629 ) ; for da agonists ,  = 4.657 ( 95% ci , 1.5527.762 ; p = 0.003 ) ; for l - dopa and da agonists ,  = 4.561 ( 95% ci , 1.9737.147 ; p = 0.001 ) . the nurr1 mrna relative level was made using the following formulas : nurr1 mrna level = 2.973 + 4.551  da agonists + 4.398  l - dopa and da agonists , or nurr1 mrna level = 4.545 + 4.657  da agonists + 4.561  l - dopa and da agonists . according to different drug treatments , the pd patients were divided into 4 subgroups . as shown in figure 1 , the relative levels of nurr1 mrna in de novo subgroup and hcs were 2.38  0.42 versus 3.18  0.42 , respectively ( p = 0.307 ) , a slight decrease but no statistical significance in de novo subgroup without any da agonists , l - dopa or other anti - pd drugs . the relative level of nurr1 mrna in l - dopa pd group was 3.37  0.57 versus 3.18  0.42 in hc ( p = 0.841 ) , which showed no statistical significance . however , the relative nurr1 mrna level in da agonist subgroup was 7.52  1.82 , significant higher than those both in de novo group ( p < 0.001 ) and hc ( p = 0.001 ) . the nurr1 mrna level in da agonist and l - dopa subgroup was up to 7.37  1.07 , which was significantly increased versus de novo subgroup or l - dopa subgroup ( p < 0.001 ) . there were no significant differences of nurr1 mrna level between da agonist and da agonist and l - dopa subgroups . scatter plots of nurr1 mrna relative levels in peripheral blood mononuclear cells of subgroups of pd patients . fluorescent reading from real - time polymerase chain reaction was quantitatively analyzed by determining the difference of ct ( delta ct ) between ct of nurr1 and internal control . hc : healthy control ; pd : parkinson 's disease ; de novo : pd without any anti - pd treatment ; da agonists : pd treated with da agonists ; l - dopa : pd treated with l - dopa ; l - dopa and da agonists : pd treated with l - dopa and da agonists ; da : dopaminergic . in 136 recent - onset cases , 25 were treated with l - dopa and 19 were treated with da agonists . the nurr1 mrna level in 25 patients was 7.87  1.70 before l - dopa treatment , and 7.59  1.59 after l - dopa taken , but no significant difference ( p = 0.905 ) . the nurr1 mrna levels in 19 patients before and after da agonist treatment were 5.92  1.42 and 8.47  2.52 , respectively , indicating a slight but nonsignificant increase after da agonist treatment ( p = 0.066 ) . scatter plots of nurr1 mrna relative levels in peripheral blood mononuclear cells before and after treatment of l - dopa ( a ) ( n = 25 ) or da agonist ( b ) ( n = 19 ) in de novo subgroup . there were no obvious changes before and after l - dopa treatment neither for the whole group nor for each individual , while there were three individuals showed obvious increase of nurr1 mrna levels though there was no significance between before and after da agonist treatment as a whole group . da : dopaminergic . to further explore whether da agonists have effect on nurr1 expression , we measured the nurr1 mrna level in pbmc from 10 healthy volunteers with 10 mol / l pramipexole [ figure 3 ] . compared with control , the nurr1 mrna relative levels in pbmc by da agonist were significantly increased to 199.61  13.17% , 171.75  11.33% , 173.16  11.42% in 2 , 4 and 8 h , respectively ( p < 0.001 ) . in vitro experiment , the nurr1 mrna levels of pbmc treated by da agonist for 12 h and 24 h were 95.30  6.26% and 104.60  4.32% , respectively , and there were no significant differences compared with control ( p > 0.05 ) , which was in accord with the half - life of 812 h. nurr1 mrna relative levels in vitro measured by real - time polymerase chain reaction treated with pramipexole . peripheral blood mononuclear cells from health volunteers were treated with 10 mol / l pramipexole for 024 h. glyceraldehyde-3-phosphate dehydrogenase was monitored as internal control . all experiments were performed at least three independent times ( * p < 0.001 ) . nurr1 is highly expressed in the developing and adult ventral midbrain and required for the acquisition and maintenance of the da phenotype in nigrostriatal neurons . it has been reported that besides central nervous system , nurr1 is also an expression in many tissues including bone , endothelial cells , and pbmc . the key point for clinical evacuation of nurr1 is whether the alteration of nurr1expression is secondary to anti - pd drug effects . using rt - pcr , we analyzed the nurr1 mrna level in pbmc from 362 sporadic pd patients and 193 hcs from southern china , and found that the nurr1 expression level in de novo subgroup with a tendency of nonsignificant decrease compared to hcs , but a statistically significant increase in both da agonist or da agonist and l - dopa subgroups , which indicated that da agonists may exert an up - regulation effect on the expression of nurr1 in pbmc . as a transcription factors , the expression of nurr1 can be affected by many factors including growth factors , neurotransmitters , and drugs . for example , injection of 6-hydroxydopamine into the striatum produced an increase in the number of cells expressing nurr1 in both substantia nigra compacta ( snc ) and substantia nigra reticulate . similar to our data , a series of evidence indicated that da receptor agonists may play an active role on the nurr1 expression . for instance , ropinirole has been documented to prevent the progression of pd in nurr1 deficient mouse , and pan et al . reported that sh - sy5y cells treated with d3 receptor agonist pramipexole enhanced the expression of nurr1 mrna and protein in vitro . studies on postmortem brains have been found that an age - related decline of da phenotypic markers was associated with down - regulation of nurr1 expression in human sn . reported the optical density of nurr1 immunofluorescence was significantly decreased in nigral neurons containing -synuclein - immunoreactive inclusions in pd patients . in addition , recent studies showed a significant decreased of nurr1 mrna in snc of d2 dopamine receptor / mice ( d2r ) . however , our data showed that the expression of nurr1 mrna in pbmc was affected by drugs like da agonist , but was no significant correlation with the disease severity ( h and y scale ) or age of onset of pd . for further exploring the pattern of nurr1 expression in vitro , we found that nurr1 mrna relative levels in pbmc increased significantly after treated with 10 mol / l pramipexole for 2 , 4 , and 8 h , which in accordance with our clinical investigation from blood cells of pd patients , demonstrating da agonist plays an active effect on nurr1 gene in pbmc . reported that levels of nurr1 mrna and protein in sh - sy5y increased after pramipexole treatment , indicating that the biological pattern of nurr1 expression in pbmc might be different from the changes in degeneration of da neurons in midbrain . in summary , we observed that nurr1 mrna level in pbmc from our pd patients was significantly influenced by antiparkinsonism drugs da agonists but not l - dopa , and such effect may contribute to da agonists - mediated neuroprotection on da neurons . furthermore , we found a slight insignificant reduction of nurr1 level in our de novo pd patients , which might present a correlation with the disease . a larger sample of de novo pd patients may be needed to evaluate whether reduced expression of nurr1 in pbmc can be used as a biomarker for early pd .