BACKGROUND
parkinson’s disease  is a progressive neurological disorder mainly due to the degeneration of dopaminergic neuronal cells in the substantia nigra pars compacta  <cit> .
the precursor of dopamine, l- <dig> -dihydroxyphenylalanine , is the most effective known therapy for controlling the motor symptoms in pd such as slowness, rigidity, resting tremor, and postural instability  <cit> .
however, chronic l-dopa administration results in a loss of drug efficacy and irreversible adverse effects, and also leads to the development of sever motor fluctuations such as l-dopa-induced-dyskinesia   <cit> .
typically, dyskinesia can occur in association with high concentrations of l-dopa in the brain and maximum improvement in the motor responses  <cit> .
the most common type of dyskinesia called peak-dose dyskinesia, occurs throughout the “on” time period to response of l-dopa administration.
although the mechanisms underlying the development and expression of lid are complex, the development of lid is associated with changes in the gene and protein expressions of ∆fosb and extracellular signal-regulated kinases  in nigral dopamine cell loss  <cit> .
lid increases striatal levels of ∆fosb and erk1/ <dig>  <cit> .
chronic l-dopa administration in 6-hydroxydopamine -lesioned rats also induces fosb-like protein ∆fosb in the dopamine-denervated striatum that develops dyskinesias  <cit> .
∆fosb protein is induced by l-dopa administration in the striatum of 6-ohda-lesioned rats  <cit> .
in addition, increased erk1/ <dig> phosphorylation by dopamine agonists in 6-ohda-lesioned striatal neurons is a possible mechanism of lid  <cit> .
erk1/ <dig> phosphorylation correlates with increased ∆fosb and dyskinesia in the depleted striatum of 6-ohda-lesioned rats  <cit> .
therefore, ∆fosb expression and erk1/ <dig> phosphorylation in the striatum are implicated in the development of lid as biological molecular markers  <cit> .
gynostemma pentaphyllum makino   is a well-known herbal medicinal plant in southeast asia.
gp contains approximately  <dig> dammarane-type glycosides , flavonoids, polysaccharides, amino acids, vitamins, and some essential elements  <cit> .
recently, gps has been shown to have anxiolytic effects on affective disorders and neuroprotective effects in 1-methyl-4-phenyl- <dig> , <dig> -tetrahydropyridine -lesioned mouse model of pd  <cit> .
in addition, ethanol extract from gp  protects the dopaminergic neurons in 6-ohda-lesioned rat model of pd  <cit> .
gp-ex also has anti-stress effects in mice  <cit> .
although gps has therapeutic benefits for several neurodegenerative diseases including pd, the effects of gps on the development of lid have not been identified yet.
in this study, therefore, we examined the effects of gps and gp-ex on lid using the rat model of lid.
after treatments with gps and gp-ex in l-dopa-administered 6-ohda-lesioned rats, lid was analyzed by using the score of abnormal involuntary movements  , contralateral rotational behaviors, and the biomarker activities of ∆fosb expression and erk1/ <dig> phosphorylation.
RESULTS
effects of gps and gp-ex on lid
in order to measure the effects of gps and gp-ex on lid, the treatment schedule including the behavioral and biochemical analyses is presented in figure  <dig>  the body aims score  and locomotive aims score using the different dyskinesia subtypes  were analyzed separately.
the daily repeated administration of l-dopa  induced an increase over time in the body aims score in 6-ohda-lesioned rats.
lid by the body aims score was observed at day- <dig> and continued to increase at day- <dig>  reaching its maximum at day- <dig> and day- <dig> .
the body aims score of lid was slightly decreased at day- <dig> and day- <dig>  which was comparable with the previously reported results  <cit> .
in this study, the behavioral tests and biochemical analyses were examined for 22 days.figure  <dig> experimental design.
rats  were divided into five groups  and pd models were established by 6-ohda lesion .
three weeks after the 6-ohda lesion, the apomorphine-induced rotational test was carried out to assess the efficacy of the 6-ohda lesion.
l-dopa  and benserazide  treatment was started 6 weeks after the 6-ohda lesion once a day for 22 days.
either gps  or gp-ex  was given 30 min prior to l-dopa treatment once a day for 22 days.
the aims were scored for 1 min every 20 min for total 180 min after l-dopa treatment on the indicated day.
on day- <dig>  after behavioral measurements, the animals were sacrificed for biochemical analyses.
p <  <dig>  compared with saline-treated group at each treatment day; #
p <  <dig>  compared with l-dopa alone-treated group at each treatment day ; †
p <  <dig>  compared with the score of day- <dig> , b: §
p <  <dig>  compared with saline-treated group; §§
p <  <dig>  compared with l-dopa alone-treated group .
the body aims score and locomotive aims score were not affected in the sham-operated rats, which had unaffected aims scores.
however, treatment with gps  or gp-ex  in 6-ohda-lesioned rats treated with l-dopa for 22 days displayed much less severe dyskinesia , compared with 6-ohda-lesioned rats treated with l-dopa .
next, the effects of gps and gp-ex on the different dyskinesia subtypes such as the body aims score  and locomotive aims score were analyzed.
the axial, limb and orolingual aims score was increased by the daily repeated administration of l-dopa  for 22 days in 6-ohda-lesioned rats .
in contrast, daily repeated treatments with gps  or gp-ex  prior to l-dopa administration in 6-ohda-lesioned rats treated with l-dopa significantly attenuated the development of l-dopa-induced body aims .figure  <dig> effects of gps and gp-ex on l-dopa-induced aims in 6-ohda-lesioned rats.
l-dopa  and benserazide  treatment was started 6 weeks after the 6-ohda lesion once a day for 22 days.
either gps  or gp-ex  was given 30 min prior to l-dopa treatment once a day for 22 days.
the aims were scored for 1 min every 20 min for total 180 min after l-dopa treatment on the indicated day according to the description in the materials and methods section.
a: axial aims; b: limb aims; c: orolingual aims; d: locomotive aims.
the results are expressed as mean ± s.e.m.
for 8– <dig> animals/group.
–■–, saline treatment; −□–, l-dopa; −▲–, l-dopa + gp-ex ; −◆–, l-dopa + gps ; −○–, l-dopa + gps .
*
p <  <dig>  compared with saline-treated group at each treatment day; #
p <  <dig>  compared with l-dopa alone-treated group at each treatment day ; †
p <  <dig>  compared with the score of day- <dig> .
in addition, the locomotive aims score was not affected by repeated treatments with l-dopa  in 6-ohda-lesioned rats.
treatment with gps  or gp-ex  in 6-ohda-lesioned rats treated with l-dopa also did not reduce the locomotive aims score, compared with gps- or gp-ex-untreated group .
effects of gps and gp-ex on contralateral rotational behavior
gps  and gp-ex  were co-treated with l-dopa administration of 6-ohda-lesioned rats to determine their effects on rotational behavior.
l-dopa administration  in 6-ohda-lesioned rats produced a significant increase in contralateral turns, compared with saline-treated 6-ohda-lesioned group .
treatment with gps  or gp-ex  for 22 days in 6-ohda-lesioned rats treated with l-dopa also slightly increased the number of contralateral turns, compared with 6-ohda-lesioned rats treated with l-dopa, but it was not significant .
in addition, a slight enhancement of contralateral rotations on the anti-parkinsonian effects of l-dopa was observed in gps -treated group .
however, no significant interaction between l-dopa and gps or gp-ex was observed .figure  <dig> effects of gps and gp-ex on contralateral rotation behavior in 6-ohda-lesioned rats.
l-dopa  and benserazide  treatment was started 6 weeks after the 6-ohda-lesion once a day for 22 days.
gps  and gp-ex  were given 30 min prior to l-dopa treatment once a day for 22 days.
the contralateral rotation behavior was monitored for 1 h on the indicated day.
the results are expressed as mean ± s.e.m.
for 8– <dig> animals/group.
–■–, saline treatment; −□–, l-dopa; −▲–, l-dopa + gp-ex ; −◆–, l-dopa + gps ; −○–, l-dopa + gps .
*
p <  <dig>  compared with saline-treated group at each treatment day ; †
p <  <dig>  compared with the score of day- <dig> .
effects of gps and gp-ex on Δfosb expression
degeneration of dopaminergic neurons by both 6-ohda and l-dopa treatment induced an overall increase in striatal levels of ∆fosb, which was measured by western blot .
l-dopa  administration resulted in a  <dig> -fold  increase in expression of ∆fosb in 6-ohda-lesioned rats, compared with 6-ohda-lesioned rats treated with saline .
however, co-treatment with gps  prior to l-dopa administration decreased the expression of ∆fosb to  <dig> - and  <dig> -fold , compared with 6-ohda-lesioned rats treated with l-dopa .
co-treatment with gp-ex  prior to l-dopa administration also decreased the expression of ∆fosb , compared with 6-ohda-lesioned rats treated with l-dopa .figure  <dig> effects of gps and gp-ex on l-dopa-induced expression of ∆fosb in 6-ohda-lesioned rats.
∆fosb expression was evaluated by western blotting of proteins extracted from the 6-ohda-lesioned striatum 1 h after the final l-dopa treatment.
immunoblot images were detected by antibodies against ∆fosb and β-actin using western blotting analysis.
values of the relative density ratios of ∆fosb expression, which are normalized by β-actin, are expressed in arbitrary units as compared with saline-treated group or l-dopa alone-treated group.
the position of molecular size markers is indicated as kda.
the results are expressed as mean ± s.e.m.
for 8– <dig> animals/group.
*
p <  <dig>  compared with saline-treated group; #
p <  <dig>  compared with l-dopa alone-treated group .
effects of gps and gp-ex on erk1/ <dig> phosphorylation
l-dopa  administration resulted in  <dig> -fold  increase in erk1/ <dig> phosphorylation in 6-ohda-lesioned rats, compared with 6-ohda-lesioned rats treated with saline .
co-treatment with gps  prior to l-dopa administration  reduced erk1/ <dig> phosphorylation  respectively, compared with 6-ohda-lesioned rats treated with l-dopa .
co-treatment with gp-ex  prior to l-dopa administration  also decreased erk1/ <dig> phosphorylation , compared with 6-ohda-lesioned rats treated with l-dopa .figure  <dig> effects of gps and gp-ex on l-dopa-induced phosphorylation of erk1/ <dig> in 6-ohda-lesioned rats.
erk1/ <dig> phosphorylation  was evaluated by western blotting of proteins extracted from the 6-ohda-lesioned striatum 1 h after the final l-dopa treatment.
immunoblot images were detected by antibodies against phospho-erk1/ <dig>  erk1/ <dig> and β-actin using western blotting analysis.
values of the relative density ratios of p-erk1/2/erk1/ <dig> are normalized and expressed in arbitrary units as compared with saline-treated group or l-dopa alone-treated group.
the position of molecular size markers is indicated as kda.
the results are expressed as mean ± s.e.m.
for 8– <dig> animals/group.
*p <  <dig>  compared with saline-treated group; #
p <  <dig>  compared with l-dopa alone-treated group .
discussion
gps and gp-ex showed anxiolytic effects on affective disorders in mptp-lesioned mouse model of pd with or without l-dopa administration  <cit> .
gps  showed protective effects in mptp-lesioned mouse model of pd  <cit> .
gp-ex also has protective effects on neurotoxicity by modulating th neuronal cell death and dopamine levels in 6-ohda-lesioned rat model of pd  <cit> .
on the basis of these results, we investigated the effects of gps and gp-ex on the developments of lid in 6-ohda-lesioned rat model of pd.
the body aims score such as axial, limb and orolingual aims score is increased in lid in 6-ohda-lesioned animal model of pd  <cit> .
however, the locomotive aims score does not necessarily provide a behavioral correlate of lid  <cit> .
in this study, lid expression was significantly increased by chronic l-dopa administration for 22 days in 6-ohda-lesioned rats .
however, co-treatment with gps  or gp-ex  prior to l-dopa administration significantly attenuated the development of lid as determined by body aims score .
in contrast, the locomotive aims score was not altered by co-treatment with gps  or gp-ex  prior to l-dopa administration .
l-dopa treatment increased the number of contralateral rotations in 6-ohda-lesioned rats, indicating behavior sensitization.
the contralateral rotations are indicative of anti-parkinsonian efficacy.
however, it is also suggested that the rotational behaviors are a dyskinesia symptom  <cit> .
in this study, treatment with gps  or gp-ex  in 6-ohda-lesioned rats administered with l-dopa slightly increased the contralateral rotations, but it was not significant , suggesting that gps  and gp-ex  treatment did not produce a significant behavior sensitization.
furthermore, these results indicate that the anti-dyskinetic efficacy of gps and gp-ex is not due to an attenuation of l-dopa efficacy.
the striatal ∆fosb is induced in the development of lid in pd rat models  <cit> .
6-ohda-lesioned rats and l-dopa administration in saline-lesioned rats do not affect ∆fosb  <cit> .
in addition, the phosphorylated levels of erk1/ <dig> are not affected by saline treatment in 6-ohda-lesioned rats  <cit> .
the pronounced phosphorylation of erk1/ <dig> in the striatum of 6-ohda-lesioned rats provides a molecular counterpart to the induction of aims by l-dopa administration  <cit> .
the present study showed the significant ∆fosb expression and erk1/ <dig> phosphorylation after l-dopa administration in the striatum of 6-ohda-lesioned rats .
however, the increased ∆fosb expression after l-dopa administration in the striatum of 6-ohda-lesioned rats was reduced by treatment with either gps  or gp-ex  prior to l-dopa administration .
gps  and gp-ex  also decreased erk1/ <dig> phosphorylation in 6-ohda-lesioned rats after l-dopa administration.
these results suggest that the effects of gps and gp-ex on the development of lid are mediated by the modulation of ∆fosb expression and erk1/ <dig> phosphorylation in 6-ohda-lesioned rats.
it is reported that lid affects up to 80% of pd patients after l-dopa administration for 5– <dig> years and some of pd patients have to stop the therapy due to severe lid  <cit> , which is difficult to treat.
the pathogenesis of lid remains incompletely understood presently.
however, it is known that dyskinesias appear only after dopaminergic therapy and the presence of dopaminergic cell loss in the substantia nigra  <cit> .
furthermore, there is evidence that supersensitivity in dopamine-depleted striatum results from pulsatile stimulation of postsynaptic dopamine receptor  <cit> .
the increases in ∆fosb expression after l-dopa administration have been shown to be associated with the supersensitive responses to l-dopa induced by dopaminergic stimulation in dopamine depleted striatum  <cit> .
the sustained activation of erk1/ <dig> by l-dopa administration also reflects the supersensitivity of dopamine receptor-dependent signaling in dopamine-depleted striatum  <cit> .
in addition, l-dopa induces ∆fosb expression in striatal neurons by involving d <dig> receptors and erk1/ <dig> phosphorylation  <cit> .
in 6-ohda-lesioned striatum, both d <dig> and d <dig> receptor agonists have been reported to significantly induce erk1/ <dig> activation  <cit> .
these results suggest that changes in ∆fosb expression and erk signaling system are associated with the supersensitive responses to dopaminergic stimulation, which is implicated in lid in dopamine-depleted striatum.
recently, the sustained erk1/ <dig> phosphorylation induced by l-dopa has been shown to possibly lead to dopaminergic neuronal cell death in pc <dig> cells  <cit> , and gps has shown protective effects on 1-methyl-4-phenylpyridinium -induced oxidative injury of dopaminergic neurons in primary culture  <cit> .
therefore, the protective effects of gps and gp-ex on dopaminergic neuronal cells can be involved in the inhibition of the development of lid.
gps is a potent free radical scavenger, which strongly increases the superoxide dismutase activity  <cit> .
gps shows protective effects in aortic endothelial cells against oxidative damage  <cit>  and on oxidative stress induced by glutamate-induced neurotoxicity  <cit> .
gp-ex also has been reported to have anti-stress and immunomodulatory effects in mice  <cit> .
gp-ex has a protective function against chronic stress by modulation of c-fos expression  <cit> .
furthermore, l-dopa increases nitric oxide production in the striatum which is associated with pd  <cit> .
neuronal nitric oxide synthase inhibition attenuates the expression of lid  <cit> .
daily repeated l-dopa administration also increases the nitric oxide generation through activation of neural nitric oxide synthase and this nitric oxide develops lid through a post-synaptic mechanism by the accumulation of ∆fosb  <cit> .
in contrast, it is suggested that the development of lid is closely associated with progression of pre-synaptic dopaminergic neurodegeneration in the substantia nigra  <cit> .
it is therefore suggested that the ameliorating effects of gps and gp-ex on lid might be mediated by scavenging the ros formation in the striatum of 6-ohda-lesioned rats.
however, whether the functions of gps and gp-ex on reducing Δfosb levels are involved in the post-synaptic mechanism needs to be studied further.
the dose of gps  and gp-ex  treatments does not exhibit adverse effects, such as weight loss, diarrhea, vomiting, and death  <cit> .
the ld <dig> values of total gps are 755–838 mg/kg  and  <dig>  in mice  <cit> , indicating that gps and gp-ex are low toxicity therapeutic agents.
CONCLUSIONS
the present study demonstrated that gps  and gp-ex  not only attenuated the development of lid, but also reduced both ∆fosb expression and erk1/ <dig> phosphorylation in 6-ohda-lesioned rats treated with chronic l-dopa.
gps and gp-ex also had no negative effects on the anti-parkinsonian efficacy of l-dopa, which was defined by the contralateral rotational tests.
it is therefore proposed that gps and gp-ex as very low toxic agents can be helpful in preventing the l-dopa-induced adverse or toxic effects for pd patients, as well as in slowing down the progression of lid.
the clinical applications need to be studied further.

 parkinson’s disease is a progressive neurological disorder mainly due to the degeneration of dopaminergic neuronal cells in the substantia nigra pars compacta. the precursor of dopamine, l- -dihydroxyphenylalanine, is the most effective known therapy for controlling the motor symptoms in pd such as slowness, rigidity, resting tremor, and postural instability. however, chronic l-dopa administration results in a loss of drug efficacy and irreversible adverse effects, and also leads to the development of sever motor fluctuations such as l-dopa-induced-dyskinesia. typically, dyskinesia can occur in association with high concentrations of l-dopa in the brain and maximum improvement in the motor responses. the most common type of dyskinesia called peak-dose dyskinesia, occurs throughout the “on” time period to response of l-dopa administration. although the mechanisms underlying the development and expression of lid are complex, the development of lid is associated with changes in the gene and protein expressions of ∆fosb and extracellular signal-regulated kinases in nigral dopamine cell loss. lid increases striatal levels of ∆fosb and erk1/. chronic l-dopa administration in 6-hydroxydopamine -lesioned rats also induces fosb-like protein ∆fosb in the dopamine-denervated striatum that develops dyskinesias. ∆fosb protein is induced by l-dopa administration in the striatum of 6-ohda-lesioned rats. in addition, increased erk1/ phosphorylation by dopamine agonists in 6-ohda-lesioned striatal neurons is a possible mechanism of lid. erk1/ phosphorylation correlates with increased ∆fosb and dyskinesia in the depleted striatum of 6-ohda-lesioned rats. therefore, ∆fosb expression and erk1/ phosphorylation in the striatum are implicated in the development of lid as biological molecular markers. gynostemma pentaphyllum makino is a well-known herbal medicinal plant in southeast asia. gp contains approximately dammarane-type glycosides, flavonoids, polysaccharides, amino acids, vitamins, and some essential elements. recently, gps has been shown to have anxiolytic effects on affective disorders and neuroprotective effects in 1-methyl-4-phenyl-, -tetrahydropyridine -lesioned mouse model of pd. in addition, ethanol extract from gp protects the dopaminergic neurons in 6-ohda-lesioned rat model of pd. gp-ex also has anti-stress effects in mice. although gps has therapeutic benefits for several neurodegenerative diseases including pd, the effects of gps on the development of lid have not been identified yet. in this study, therefore, we examined the effects of gps and gp-ex on lid using the rat model of lid. after treatments with gps and gp-ex in l-dopa-administered 6-ohda-lesioned rats, lid was analyzed by using the score of abnormal involuntary movements, contralateral rotational behaviors, and the biomarker activities of ∆fosb expression and erk1/ phosphorylation.  effects of gps and gp-ex on lid in order to measure the effects of gps and gp-ex on lid, the treatment schedule including the behavioral and biochemical analyses is presented in figure  the body aims score and locomotive aims score using the different dyskinesia subtypes were analyzed separately. the daily repeated administration of l-dopa induced an increase over time in the body aims score in 6-ohda-lesioned rats. lid by the body aims score was observed at day- and continued to increase at day- reaching its maximum at day- and day-. the body aims score of lid was slightly decreased at day- and day- which was comparable with the previously reported results. in this study, the behavioral tests and biochemical analyses were examined for 22 days.figure experimental design. rats were divided into five groups and pd models were established by 6-ohda lesion. three weeks after the 6-ohda lesion, the apomorphine-induced rotational test was carried out to assess the efficacy of the 6-ohda lesion. l-dopa and benserazide treatment was started 6 weeks after the 6-ohda lesion once a day for 22 days. either gps or gp-ex was given 30 min prior to l-dopa treatment once a day for 22 days. the aims were scored for 1 min every 20 min for total 180 min after l-dopa treatment on the indicated day. on day- after behavioral measurements, the animals were sacrificed for biochemical analyses. p <  compared with saline-treated group at each treatment day; # p <  compared with l-dopa alone-treated group at each treatment day; † p <  compared with the score of day-, b: § p <  compared with saline-treated group; §§ p <  compared with l-dopa alone-treated group. the body aims score and locomotive aims score were not affected in the sham-operated rats, which had unaffected aims scores. however, treatment with gps or gp-ex in 6-ohda-lesioned rats treated with l-dopa for 22 days displayed much less severe dyskinesia, compared with 6-ohda-lesioned rats treated with l-dopa. next, the effects of gps and gp-ex on the different dyskinesia subtypes such as the body aims score and locomotive aims score were analyzed. the axial, limb and orolingual aims score was increased by the daily repeated administration of l-dopa for 22 days in 6-ohda-lesioned rats. in contrast, daily repeated treatments with gps or gp-ex prior to l-dopa administration in 6-ohda-lesioned rats treated with l-dopa significantly attenuated the development of l-dopa-induced body aims.figure effects of gps and gp-ex on l-dopa-induced aims in 6-ohda-lesioned rats. l-dopa and benserazide treatment was started 6 weeks after the 6-ohda lesion once a day for 22 days. either gps or gp-ex was given 30 min prior to l-dopa treatment once a day for 22 days. the aims were scored for 1 min every 20 min for total 180 min after l-dopa treatment on the indicated day according to the description in the materials and methods section. a: axial aims; b: limb aims; c: orolingual aims; d: locomotive aims. the results are expressed as mean ± s.e.m. for 8– animals/group. –■–, saline treatment; −□–, l-dopa; −▲–, l-dopa + gp-ex; −◆–, l-dopa + gps; −○–, l-dopa + gps. * p <  compared with saline-treated group at each treatment day; # p <  compared with l-dopa alone-treated group at each treatment day; † p <  compared with the score of day-. in addition, the locomotive aims score was not affected by repeated treatments with l-dopa in 6-ohda-lesioned rats. treatment with gps or gp-ex in 6-ohda-lesioned rats treated with l-dopa also did not reduce the locomotive aims score, compared with gps- or gp-ex-untreated group. effects of gps and gp-ex on contralateral rotational behavior gps and gp-ex were co-treated with l-dopa administration of 6-ohda-lesioned rats to determine their effects on rotational behavior. l-dopa administration in 6-ohda-lesioned rats produced a significant increase in contralateral turns, compared with saline-treated 6-ohda-lesioned group. treatment with gps or gp-ex for 22 days in 6-ohda-lesioned rats treated with l-dopa also slightly increased the number of contralateral turns, compared with 6-ohda-lesioned rats treated with l-dopa, but it was not significant. in addition, a slight enhancement of contralateral rotations on the anti-parkinsonian effects of l-dopa was observed in gps -treated group. however, no significant interaction between l-dopa and gps or gp-ex was observed.figure effects of gps and gp-ex on contralateral rotation behavior in 6-ohda-lesioned rats. l-dopa and benserazide treatment was started 6 weeks after the 6-ohda-lesion once a day for 22 days. gps and gp-ex were given 30 min prior to l-dopa treatment once a day for 22 days. the contralateral rotation behavior was monitored for 1 h on the indicated day. the results are expressed as mean ± s.e.m. for 8– animals/group. –■–, saline treatment; −□–, l-dopa; −▲–, l-dopa + gp-ex; −◆–, l-dopa + gps; −○–, l-dopa + gps. * p <  compared with saline-treated group at each treatment day; † p <  compared with the score of day-. effects of gps and gp-ex on Δfosb expression degeneration of dopaminergic neurons by both 6-ohda and l-dopa treatment induced an overall increase in striatal levels of ∆fosb, which was measured by western blot. l-dopa administration resulted in a -fold increase in expression of ∆fosb in 6-ohda-lesioned rats, compared with 6-ohda-lesioned rats treated with saline. however, co-treatment with gps prior to l-dopa administration decreased the expression of ∆fosb to - and -fold, compared with 6-ohda-lesioned rats treated with l-dopa. co-treatment with gp-ex prior to l-dopa administration also decreased the expression of ∆fosb, compared with 6-ohda-lesioned rats treated with l-dopa.figure effects of gps and gp-ex on l-dopa-induced expression of ∆fosb in 6-ohda-lesioned rats. ∆fosb expression was evaluated by western blotting of proteins extracted from the 6-ohda-lesioned striatum 1 h after the final l-dopa treatment. immunoblot images were detected by antibodies against ∆fosb and β-actin using western blotting analysis. values of the relative density ratios of ∆fosb expression, which are normalized by β-actin, are expressed in arbitrary units as compared with saline-treated group or l-dopa alone-treated group. the position of molecular size markers is indicated as kda. the results are expressed as mean ± s.e.m. for 8– animals/group. * p <  compared with saline-treated group; # p <  compared with l-dopa alone-treated group. effects of gps and gp-ex on erk1/ phosphorylation l-dopa administration resulted in -fold increase in erk1/ phosphorylation in 6-ohda-lesioned rats, compared with 6-ohda-lesioned rats treated with saline. co-treatment with gps prior to l-dopa administration reduced erk1/ phosphorylation respectively, compared with 6-ohda-lesioned rats treated with l-dopa. co-treatment with gp-ex prior to l-dopa administration also decreased erk1/ phosphorylation, compared with 6-ohda-lesioned rats treated with l-dopa.figure effects of gps and gp-ex on l-dopa-induced phosphorylation of erk1/ in 6-ohda-lesioned rats. erk1/ phosphorylation was evaluated by western blotting of proteins extracted from the 6-ohda-lesioned striatum 1 h after the final l-dopa treatment. immunoblot images were detected by antibodies against phospho-erk1/ erk1/ and β-actin using western blotting analysis. values of the relative density ratios of p-erk1/2/erk1/ are normalized and expressed in arbitrary units as compared with saline-treated group or l-dopa alone-treated group. the position of molecular size markers is indicated as kda. the results are expressed as mean ± s.e.m. for 8– animals/group. *p <  compared with saline-treated group; # p <  compared with l-dopa alone-treated group. discussion gps and gp-ex showed anxiolytic effects on affective disorders in mptp-lesioned mouse model of pd with or without l-dopa administration. gps showed protective effects in mptp-lesioned mouse model of pd. gp-ex also has protective effects on neurotoxicity by modulating th neuronal cell death and dopamine levels in 6-ohda-lesioned rat model of pd. on the basis of these results, we investigated the effects of gps and gp-ex on the developments of lid in 6-ohda-lesioned rat model of pd. the body aims score such as axial, limb and orolingual aims score is increased in lid in 6-ohda-lesioned animal model of pd. however, the locomotive aims score does not necessarily provide a behavioral correlate of lid. in this study, lid expression was significantly increased by chronic l-dopa administration for 22 days in 6-ohda-lesioned rats. however, co-treatment with gps or gp-ex prior to l-dopa administration significantly attenuated the development of lid as determined by body aims score. in contrast, the locomotive aims score was not altered by co-treatment with gps or gp-ex prior to l-dopa administration. l-dopa treatment increased the number of contralateral rotations in 6-ohda-lesioned rats, indicating behavior sensitization. the contralateral rotations are indicative of anti-parkinsonian efficacy. however, it is also suggested that the rotational behaviors are a dyskinesia symptom. in this study, treatment with gps or gp-ex in 6-ohda-lesioned rats administered with l-dopa slightly increased the contralateral rotations, but it was not significant, suggesting that gps and gp-ex treatment did not produce a significant behavior sensitization. furthermore, these results indicate that the anti-dyskinetic efficacy of gps and gp-ex is not due to an attenuation of l-dopa efficacy. the striatal ∆fosb is induced in the development of lid in pd rat models. 6-ohda-lesioned rats and l-dopa administration in saline-lesioned rats do not affect ∆fosb. in addition, the phosphorylated levels of erk1/ are not affected by saline treatment in 6-ohda-lesioned rats. the pronounced phosphorylation of erk1/ in the striatum of 6-ohda-lesioned rats provides a molecular counterpart to the induction of aims by l-dopa administration. the present study showed the significant ∆fosb expression and erk1/ phosphorylation after l-dopa administration in the striatum of 6-ohda-lesioned rats. however, the increased ∆fosb expression after l-dopa administration in the striatum of 6-ohda-lesioned rats was reduced by treatment with either gps or gp-ex prior to l-dopa administration. gps and gp-ex also decreased erk1/ phosphorylation in 6-ohda-lesioned rats after l-dopa administration. these results suggest that the effects of gps and gp-ex on the development of lid are mediated by the modulation of ∆fosb expression and erk1/ phosphorylation in 6-ohda-lesioned rats. it is reported that lid affects up to 80% of pd patients after l-dopa administration for 5– years and some of pd patients have to stop the therapy due to severe lid, which is difficult to treat. the pathogenesis of lid remains incompletely understood presently. however, it is known that dyskinesias appear only after dopaminergic therapy and the presence of dopaminergic cell loss in the substantia nigra. furthermore, there is evidence that supersensitivity in dopamine-depleted striatum results from pulsatile stimulation of postsynaptic dopamine receptor. the increases in ∆fosb expression after l-dopa administration have been shown to be associated with the supersensitive responses to l-dopa induced by dopaminergic stimulation in dopamine depleted striatum. the sustained activation of erk1/ by l-dopa administration also reflects the supersensitivity of dopamine receptor-dependent signaling in dopamine-depleted striatum. in addition, l-dopa induces ∆fosb expression in striatal neurons by involving d receptors and erk1/ phosphorylation. in 6-ohda-lesioned striatum, both d and d receptor agonists have been reported to significantly induce erk1/ activation. these results suggest that changes in ∆fosb expression and erk signaling system are associated with the supersensitive responses to dopaminergic stimulation, which is implicated in lid in dopamine-depleted striatum. recently, the sustained erk1/ phosphorylation induced by l-dopa has been shown to possibly lead to dopaminergic neuronal cell death in pc cells, and gps has shown protective effects on 1-methyl-4-phenylpyridinium -induced oxidative injury of dopaminergic neurons in primary culture. therefore, the protective effects of gps and gp-ex on dopaminergic neuronal cells can be involved in the inhibition of the development of lid. gps is a potent free radical scavenger, which strongly increases the superoxide dismutase activity. gps shows protective effects in aortic endothelial cells against oxidative damage and on oxidative stress induced by glutamate-induced neurotoxicity. gp-ex also has been reported to have anti-stress and immunomodulatory effects in mice. gp-ex has a protective function against chronic stress by modulation of c-fos expression. furthermore, l-dopa increases nitric oxide production in the striatum which is associated with pd. neuronal nitric oxide synthase inhibition attenuates the expression of lid. daily repeated l-dopa administration also increases the nitric oxide generation through activation of neural nitric oxide synthase and this nitric oxide develops lid through a post-synaptic mechanism by the accumulation of ∆fosb. in contrast, it is suggested that the development of lid is closely associated with progression of pre-synaptic dopaminergic neurodegeneration in the substantia nigra. it is therefore suggested that the ameliorating effects of gps and gp-ex on lid might be mediated by scavenging the ros formation in the striatum of 6-ohda-lesioned rats. however, whether the functions of gps and gp-ex on reducing Δfosb levels are involved in the post-synaptic mechanism needs to be studied further. the dose of gps and gp-ex treatments does not exhibit adverse effects, such as weight loss, diarrhea, vomiting, and death. the ld values of total gps are 755–838 mg/kg and in mice, indicating that gps and gp-ex are low toxicity therapeutic agents.  the present study demonstrated that gps and gp-ex not only attenuated the development of lid, but also reduced both ∆fosb expression and erk1/ phosphorylation in 6-ohda-lesioned rats treated with chronic l-dopa. gps and gp-ex also had no negative effects on the anti-parkinsonian efficacy of l-dopa, which was defined by the contralateral rotational tests. it is therefore proposed that gps and gp-ex as very low toxic agents can be helpful in preventing the l-dopa-induced adverse or toxic effects for pd patients, as well as in slowing down the progression of lid. the clinical applications need to be studied further. 
these results suggest that gps and gp-ex effectively attenuate the development of lid by modulating the biomarker activities of ∆fosb expression and erk1/ phosphorylation in the 6-ohda-lesioned rat model of pd. in addition, the increases in ∆fosb expression and erk1/ phosphorylation in 6-ohda-lesioned rats induced by l-dopa administration were significantly reduced by co-treatment with gps or gp-ex. daily administration of l-dopa in the 6-ohda-lesioned rat model of pd for 22 days induced expression of lid, which was determined by the body and locomotive aims scores and contralateral rotational behaviors. gypenosides and ethanol extract of gynostemma pentaphyllum show anxiolytic effects on affective disorders in 1-methyl-4-phenyl-, -tetrahydropyridine-lesioned mouse model of parkinson’s disease. the present study investigated the effects of gps and gp-ex on lid in a 6-hydroxydopamine -lesioned rat model of pd.
 gypenosides and ethanol extract of gynostemma pentaphyllum show anxiolytic effects on affective disorders in 1-methyl-4-phenyl-, -tetrahydropyridine-lesioned mouse model of parkinson’s disease. long-term administration of l- -dihydroxyphenylalanine leads to the development of severe motor side effects such as l-dopa-induced-dyskinesia in pd. the present study investigated the effects of gps and gp-ex on lid in a 6-hydroxydopamine -lesioned rat model of pd.  daily administration of l-dopa in the 6-ohda-lesioned rat model of pd for 22 days induced expression of lid, which was determined by the body and locomotive aims scores and contralateral rotational behaviors. however, co-treatments of gps or gp-ex with l-dopa significantly attenuated the development of lid without compromising the anti-parkinsonian effects of l-dopa. in addition, the increases in ∆fosb expression and erk1/ phosphorylation in 6-ohda-lesioned rats induced by l-dopa administration were significantly reduced by co-treatment with gps or gp-ex.  these results suggest that gps and gp-ex effectively attenuate the development of lid by modulating the biomarker activities of ∆fosb expression and erk1/ phosphorylation in the 6-ohda-lesioned rat model of pd. gps and gp-ex will be useful adjuvant therapeutics for lid in pd. keywords gynostemma pentaphyllum6-hydroxydopamine-lesioned ratsdyskinesiabody and locomotive aims scores∆fosberk1/2adjuvant therapeuticsissue-copyright-statement© the author 2015 