Inhibition Of α-synuclein Aggregation And Its Associated Cytotoxicity By Brazilin And Dihydromyricetin

Parkinson’s disease(PD)is a common neurodegenerative disease,with an incidence rate of 1%among people aged 55 or older worldwide.The typical pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the production of intracellular Lewy bodies.The main component of Lewy body is pre-synaptic protein αsynuclein(α-syn).α-Syn is misfolded and then aggregates to form fibers,which are finally deposited in the brain,causing different degrees of damage to brain cells,which triggers PD.Therefore,the development of highly effective aggregation inhibitors against α-syn fibrosis is an important strategy for the treatment of PD,and it is also one of the research focuses in the field of life sciences at this stage.In this paper,α-syn was first heterologously expressed in E.coli.By optimizing the expression and purification conditions of α-syn,α-syn with a purity of more than 95%was obtained,and the yield was 54.9 mg/L.Then,the aggregation characteristics of biologically expressed α-syn were verified using thioflavin T(ThT)fluorescence experiment,atomic force microscope(AFM)and MTT cytotoxicity experiment.The results show that the biologically expressed α-syn protein can aggregate to form fibers and has strong cytotoxicity.The above research proves that α-syn can be used for conventional aggregation characteristics research and screening for aggregation inhibition.Brazilin is a natural polyphenolic compound with anti-inflammatory,antioxidant and neuroprotective properties,but the inhibitory effect of brazilin on α-syn aggregation is unknown.Aiming at this key issue,this paper uses systematic biochemical,biophysical,and cellular biological experiments to study the inhibitory effect of brazilin on α-syn fibrosis.The results of tht and AFM showed that two doses of brazilin could not only inhibit the formation of α-syn fibers,but also depolymerize mature fibers into amorphous aggregates,with almost no fluorescence,and its ability to inhibit and depolymerize mature fibers was concentration dependent.In addition,the results of cytotoxicity experiments showed that brazilin can also effectively reduce the toxic effect of α-syn aggregates on nerve cells.Considering the poor stability and easy oxidation of brazilin,this paper selects another polyphenol compound,dihydromyricetin(DHM),which has better stability,and explores its effect on α-syn fibrosis.And the inhibitory effect of aggregates on cytotoxicity.The results of tht fluorescence and AFM showed that DHM could inhibit the formation of α-syn fibrils at the same time,and the same dose of DHM could destroy the mature α-syn fibrils to form amorphous aggregates,and the inhibition effect was dose-dependent.The results of cell experiments showed that DHM can effectively reduce the toxic effect of α-syn aggregates on nerve cells,and has a strong cytoprotective effect.When α-syn was coincubated with equal dose of DHM,the activity of PC 12 cells increased by 34.73 ± 3.68%with DHM.The above results indicate that brazilin and DHM are expected to be potential drugs against PD caused by α-syn aggregation,providing experimental and theoretical foundations for the development of PD therapeutic drugs.