Preventive Effect And Mechanism Of Sulforaphane On Levodopa Induced Dyskinesia In Parkinson's Disease Mice

Levodopa-induced dyskinesias(LID)is a manifestation of dyskinesia caused by long-term use of levodopa in patients with Parkinson's disease and central nervous system regulation disorders in the later stages of treatment.The regulatory network of basal ganglia in patients of LID is pathologically changed during the treatment process,especially the 5-hydroxytryptamine(5-HT)neurons.The 5-HT neurons are mainly concentrated in the raphe nucleus(RN),and the nerve fibers are widely distributed.The cell body and the peripheral can absorb the external levodopa drug and convert it into dopamine(DA).However,5-HT neurons is lack of self-regulation mechanism to control DA release and clearance.Accumulation of DA forms quinone,3,4-dihydroxybenzoic acid or hydrogen peroxide,under oxidative stress.These toxic substances destroy important key enzymes such as tryptophan hydroxylase and damage raphe nucleus.And cause 5-HT neurons death.Therefore,finding a way to protect 5-HT cells from DA metabolite damage is an important way to prevent the onset of LID.Quinone oxidoreductase 1(NQO1)is a detoxifying enzyme of active quinone compounds and their derivatives.NQO1 prevents the formation of active semiquinone radicals and ROS,and also protects cells from oxidative damage.Sulforaphane(SFN)is an important detoxification enzyme inducer.It can promote the expression of phase II detoxification enzymes by activating Nrf2 / ARE elements,which can increase the expression of NQO1.SFN is widely used in the research of neurodegenerative diseases.However,there are few studies about the effects of SFN on dyskinesia,and nearly no animal experiments.This study combined the short-term quinone toxicity experiment and the long-term dyskinesia experiment to verify the efficacy of SFN in mouse model.The overexpress NQO1 transgenic mice were also used in these experiments to explore the correlation between SFN effect and its induction of high expression of NQO1.At the same time,through the biochemical analysis of related proteins,we investigated the correlation between the LID behavior and the mechanism of 5-HT system.The experimental mice were divided into WT group,SFN(5 mg / kg)injected SF group,and two strains of transgenic mice T27 and T56 with high expression of NQO1.The experimental work and main results of this paper include:(1)Western blotting and ELISA were used to analyze the NQO1 protein expression and enzyme activity.The results showed that the expression of NQO1 protein and the activity of NQO1 enzyme in T27,T56 and SF group had been increased significantly(P< 0.05,P< 0.001).(2)The acute administration experiment of hydroquinone.The same dose of hydroquinone was given to mice by gavage.The mice of SF,T27/T56 groups survived longer.After extracting brain stem protein and liver protein,NBT method was used to detect the content of protein quinonization.The content of quinone in SF,T27 /T56 groups were significantly lower than that in WT group(P< 0.05).(3)The Parkinson's disease model was established by unilateral injury of 6-hydroxydopamine,and the model mice were treated with levodopa continuously afterward.After regular behavioral scoring statistics,the dyskinesia performance of T27 and T56 groups was significantly lower than that of WT group(P< 0.05,P< 0.01).SF group showed no significant difference at the initial stage,but a significant relief effect in the later stage of levodopa treatment(P< 0.05).Behavioral measurements show that high expression of NQO1 has a preventive effect on the development of dyskinesia during L-DOPA treatment.(4)Western blotting analysis showed that the expression of 5-HT1 AR and tryptophan hydroxylase(TPH)in SF group were significantly higher than that in WT group(P< 0.05).In T27 and T56 groups,5-HT1 AR and TPH significantly increased compared with the WT group(P< 0.001).The density of TPH positive cells were higher in SF,T27 and T56 groups(P< 0.05,P< 0.01).The results showed that high expression of NQO1 had protective effect on 5-HT neurons in the course of levodopa treatment,which provides a valuable reference for the treatment strategy of PD.In summary,sulforaphane(SFN),as an inducer of NQO1,enhances the antiquinone toxicity of neurons by promoting the expression of highly active NQO1.In animal models of dyskinesia,highly expressed NQO1 can protect 5-HT nerves from quinone damage,and has the effect of preventing the occurrence of late motor disorder in long-term treatment with levodopa(LID).