Study On The Efficacy And Mechanism Of Shouwu Prescription On Leptopa - Induced

BackgroundLevodopa (L-DOPA) is a "golden standard" drug to treat Parkinson’s disease (PD) with significant efficacy for most PD patients. However, it causes side effects and/or complications after long-term treatment. Levodopa-induced dyskinesia (LID) is one of the serious complications that generally appears during the long-term administration of L-DOPA. The prognosis and the quality of life of the PD patients are seriously affected due to high incidence and long duration of LID. These issue needs to be appropriately addressed during treatment. Further research in this disease, taking active prevention and treatment have important social significance and far-reaching scientific significance.The severity of PD is the occurrence basis of LID. Pulsed stimulation of L-DOPA has direct impacts on the development of LID. Cortex-basal ganglia-thalamo-cortical circuit is the structural basis of LID. LID is related to the imbalance between direct pathway (excited) and indirect pathway (inhibited) mediated by dopamine (DA) receptor. DA,5-HT, excitatory amino acids-Glu and inhibitory amino acid-GABA and so on are the major neurotransmitters within this circuit. They mediate the activation and inhibition of the pathway. Excitatory toxicity caused by the increasing of excitatory amino acid also cause damage to nerve cells. It is one of the common pathogenesis of neurodegenerative diseases. In the PD stage, the balance between oxidation and antioxidation in substantia nigra and striatum was broken. Excessive highly active and toxic reactive oxygen species (ROS), hydroxyl free radicals et al, are metabolized by L-dopa and DA. They deteriorate the dysfunction of dopaminergic loop. Therefore the occurrence and development of LID were the result of a variety of pathogenic factors.ShouwuFang (SWF) is composed of Shouwu(Radix Polygoni Multiflori), Lurong (cornu cervi pantotrichum), Tianma(rhizoma gastrodiax), Gouteng(ramulus uncariae cum uncis) et al. This prescription is effective in nourishing liver and kidney, strengthening tendons and bones, dispelling wind and stop fibrillation. A clinical study showed that SWF could enhance therapeutic efficacy and reduce dose of Madopar in PD patients. Previous studies also found that SWF could increase dopamine (DA) extracellular concentration in the striatum and reduce L-DOPA fluctuation in blood and extracellular fluid of brain in PD rats, indicating the possibility of SWF in intervention of LID. Therefore, we established the LID model based on PD rats to evaluate the praxiology of the model by AIM scale; brain microdialysis combined HPLC to observe the effect of SWF on extracellular neurotransmitters and metabolites in the living target organ.For in vitro study, SH-SY5Y cells damaged by 6-OHDA were used to mimic neuronal damage. Protective effect of different concentrations of TSG, Lig and VAPs on cell viability were measured by MTT assay. Then intervention effects of these effective components of SWF on the intracellular calcium overload, excitatory toxicity and oxidative stress were observed, to find the pharmacological mechanism for SWF.Method:(1) To established the PD models by unilateral perfusion of 6-OHDA in two positions in SD rats. The PD rats were administrated with Madopa in order to induce LID models. Meanwhile the rats were co-administrated with SWF in three groups with high, medium and low dose respectively for 22 days. The last administration was on the day of microdialysis. A global abnormal involuntary movement (AIM) were evaluated, which included axial dystonia, limb dyskinesia, masticatory dyskinesia and body rotation. Each section is divided into five levels according to the severity (0-4 scores). The evaluation were performed once every seven days, four times in total. Evaluation process was described as following:score was recorded once every 30 min for 120 min after gavage. AIM scores were the total scores of 4 point-in-time. The effects of SWF on behavior of rats were assessed by the AIM scores, the incidence of LID and severe LID (AIM scores> 20).(2) Microdialysis technology was used to collect samples in the extracellular fluid of free moving LID rats. The dialysate was collected for 30 min/tube for 240 min in 2 μL/min. The extracellular concentration of excitatory and inhibitory amino acid neurotransmitters (Glu, GABA et al) in striatum of free-moving rats were measured by HPLC-FLD. The metabolites of DA were detected by HPLC-ED; salicylic acid was used to capture hydroxyl free radical in the brains, which formed stable 2,3-DHBA and 2,5-DHBA and therefore can be detected by HPLC-ED.(3) SOD activity was detected by xanthine oxidase method. MDA concentration was detected using thiobarbituric acid colorimetric method. The inflammatory cytokines IL-1β, IL-6 IL-1β in the rats were detected using Elisa method.(4) The effect of TSG, Lig and VAPs on the cell viability in 6-OHDA-induced SH-SY5Y cells was observed with MTT assay. The concentration of intracellular calcium and the distribution of Nrf2 in cells were detected using laser scanning confocal microscopy. The concentration of Glu and GABA in supernatant of different-treated cells were detected by HPLC-FLD. The intracellular concentration of HO-1 was detected using Elisa method.Results(1) PD rats appeared abnormal involuntary movement after repeated administration of Madopar, including axial dystonia, neck and upper torso twisted to the damage contralateral side and contralateral body rotation, etc. SWF can reduce the incidence of LID and AIM scores. SWF-H group significantly reduced the AIM scores at D14 and D21(P<0.05).(2) SWF can reduce the increasing of extracellular concentration of excitatory amino acid (Glu, Asp)and inhibitory amino acid(GABA, Gly) in the striatum of LID rats. Compared with LID group, Asp significantly decreased at 1 time point in SWF-L group,1 time point in SWF-M group and 3 time points in SWF-H groups respectively(P<0.05 or P<0.01). GABA decreased at 1 point in SWF-L group and SWF-H group respectively(P<0.05 or P <0 .01).(3) SWF can decrease the extracellular concentration of 2,3-DHBA and 2,5-DHBA in the striatum; meanwhile significantly lower the MDA level in the blood but had little impact on SOD activity. Compared with LID group,2,3-DHBA decreased at 1 time point in SWF-M group(P<0.05).2,5-DHBA decreased at 1 time point in SWF-L group and SWF-M group respectively(P<0.05 or P<0.01). SWF significantly inhibited the increasing of MDA in blood (P<0.01).(4) SWF can significantly alleviated the fluctuation of DOPAC and HVA, reduced the increasing of extracellular 5-HIAA in the striatum of LID rats. Compared with LID group, 5-HIAA decreased at 1 time point in SWF-M group(P<0.05).(5) Although the concentration of IL-1β increased, no change in concentration of IL-6 and TNF-a in the serum of LID rats were found. SWF can inhibit the increasing of IL-1 (3 but little impact on IL-6 and TNF-a.(6) TSG, VAPs and Lig can improve the cell viability (P<0.05 or P<0.01). TSG and Lig can resisted the increasing of intracellular free calcium and the concentration of Glu and GABA in supernatant compared with 6-OHDA-treated cells(.P<0.05 or P<0.01).(7) Lig and VAPs can promote Nrf2 transference into nucleus and increased the intracellular concentration of HO-1(P<0.05).Conclusion(1) PD rats appears dyskinesia after repeated administration of L-DOPA. Although SWF had no effect on incidence of LID, it decreased the incidence of severe LID (AIM scores>20) and ameliorated the symptoms.(2) The extracellular amino acids neurotransmitters in the striatum of LID rats were unbalanced. The increasing of excitatory amino acid also induced excitatory toxicity to nerve cells. SWF can keep the harmonious in amino acid neurotransmitter, reduce the damage caused by excitatory toxicity.(3) The extracellular concentration of index of hydroxyl free radical increased in the striatum of LID rats, meanwhile the MDA increased in the serum of rats. SWF decreased the level of hydroxyl free radical and MDA, so SWF protected the body especially the target organ via anti-oxidation.(4) The extracellular DA metabolites fluctuated along the time in the striatum of LID rats. SWF can flat this fluctuation, relieve the pulsatile stimulation of the DA receptors, then alleviated the symptoms consequently.(5) No obvious change in inflammatory cytokines were observed in LID rats. SWF had little effect on inflammatory cytokines.(6) For in vitro study, TSG, Lig and VAPs, which are the active ingredient of SWF, had protective effect on exogenous neurotoxic injury by reducing calcium overload, inhibiting the excitability toxicity and antioxidant. Both of them supported theoretical foundation of SWF.