Study On Establishing Animal Model Of Parkinson’s Disease Using Silkworm,Bombyx Mori

Parkinson disease(PD)is a common neurodegenerative disease in middle-aged and elderly people.The average age of onset is about 60 years old,and the growth rate of PD ranks first among neurological diseases.With the advent of the global aging society,the treatment of this disease is in great demand,and its research has a very prominent realistic urgency.PD has a serious impact on the quality of life of patients,and its symptoms include not only dyskinesia,but also non-motor symptoms.In recent years,with the construction and application of PD animal models,breakthrough progress has been made in PD research.But so far,the etiology and pathogenesis of PD have not been fully resolved.PD modeling currently mainly includes neurotoxin induction models,transgenic models,and models combining the two.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)is a typical PD modeling agent.The PD animal model induced by MPTP drugs can show the characteristics of PD dopaminergic neuron degeneration and corresponding movement disorders.The PARK2 gene encodes an E3 ubiquitin protein ligase.Mutations in this gene can hinder protein degradation and cause protein aggregation to produce neurotoxicity.It is the most common cause of recessive genetic PD and early-onset PD.Constructing more animal models of Parkinson’s disease from different species will help explain the pathogenesis of the disease and has potential significance for screening PD therapeutic drugs.In this study,we used MPTP feeding induction and gene editing methods to knock out Bm PARK2 to establish a silkworm model of PD.This model was evaluated with levodopa.Finally,the established PD model was used to test the therapeutic effect of lycium barbarum polysaccharide on PD,and preliminarily explore whether it can be used as a candidate drug for the treatment of PD.The main research results obtained are as follows:1.Establishing PD silkworm modelBy measuring cell viability and individual body weight,it is found that 125μM MPTP has less toxic effects on Bm N cells and individual silkworms.After feeding125μM MPTP,the results of the insect behavior recorder showed that the locomotor ability of the silkworm was reduced.The DA level was measured by UPLC and found that the DA content was significantly reduced.By measuring the changes in the antioxidant enzyme activity of silkworm larvae,it was found that the oxidative damage was significantly increased,and the expression of the brain dopaminergic neuron molecular marker tyrosine hydroxylase(TH)was significantly reduced by immunofluorescence,indicating that the dopaminergic neurons injured.These characteristics are similar to the clinical symptoms of human PD patients,indicating that the PD silkworm model has been successfully established.The CRISPR/Cas9 gene editing method was used to obtain Bm PARK2 gene knockout silkworm positive individuals,and homozygous screening is currently being conducted.2.Testing PD silkworm model with levodopaThe cytotoxicity test results showed that the mixture of levodopa and carbidopa had no obvious toxic effect on Bm N cells.After adding levodopa mixture,MPTP induced a significant increase in the motor ability and DA levels of Parkinson’s disease silkworms,and slowed down the oxidative damage of PD silkworms,but there was no significant change in TH activity and protein expression levels,indicating that there was no damage to dopaminergic neurons.It shows that the levodopa mixture is only used as a precursor of dopamine to supplement the insufficient endogenous DA of the PD silkworm,and can’t reverse the damage of dopaminergic neurons.The results of this study are consistent with the results of other PD animal models,indicating that this model can be used for preliminary drug screening.3.Using PD silkworm model to test the efficacy of LBPAfter feeding lycium barbarum polysaccharide,although the TH expression of PD silkworm did not change significantly,its exercise capacity,DA level and TH activity were significantly improved,and at the same time,it slowed down the oxidative damage.It is speculated that lycium barbarum polysaccharide can delay the process of PD.In this study,Bombyx mori was used as the experimental animal,and the PD Bombyx mori model was successfully constructed by measuring the movement ability,oxidative damage degree,DA content change and dopaminergic neuron damage of the silkworm after long-term feeding MPTP.The use of levodopa to evaluate the efficacy of this model indicates that the model can be used for preliminary drug screening.Finally,this model is used to explore the efficacy of lycium barbarum polysaccharide,and it is initially found that lycium barbarum polysaccharide has a certain therapeutic effect on PD.