Neuroprotective Mechanism And Effects Of Resveratrol And Soy Isoflavone On Different Models Of Parkinson's Disease

Parkinson's disease(PD)is a common neurodegenerative disease,mainly caused by aging,environment and genetic factor.At present,little measure can be used to cure PD.LRRK2,located in number 12 chromosome of human genome,the mutation of which will cause genetic and sporadic PD.Many studies prove that after mutation of LRRK2,the encoded protein of which will improve the kinase activity and thus leading to PD.Especially,the G2019S mutation on LRRK2 Kinase domain can apparently cause neurotoxicity and the loss of dopaminergic neuron.Therefore,searching for effective LRRK2 kinase activity inhibitors will be a significant approach to treat PD.Our experiment choose resveratrol and soy isoflavone as key object,which were screened from monomer of Chinese traditional herbs,validating the inhibition effect of LRRK2 kinase activity and cure effect on PD transgenic fly,SH-SY5Y cell,and transgenic mouse.Furthermore,we will preliminary discussing the neuroprotection mechanism of resveratrol and soy isoflavone,by studying different signal pathway.Objective:Our aim is to observe the changes of PD phenotype,LRRK2 kinase activity and LRRK2 related signal pathway downstream by using LRRK2 transgenic fly models,and rotenone-reduced SH-SY5Y cell models,treated with resveratrol and soy isoflavone respectively.We also try to identify and generate different types of LRRK2 transgenic mouse models by hybridization and test crossing.Methods:1.We also established rotenone-reduced PD cell models,and transgenic PD fly models which were generated by Gal-UAS crossing system,in order to make exogenous gene LRRK2-G2019S express in dopaminergic neuron.Moreover,we try to generate LRRK2 transgenic mouse model,through hybridization of heterozygous mice which have exogenous gene LRRK2-G2019S,and hybridization of GS2 and A53T transgenic mice..Moreover,PCR technique was used to identify homozygosis GS2 transgenic mouse model and GS2-A53T double transgenic mouse model.2.SH-SY5Y cells were treated with different concentration of resveratrol and soy isoflavone,respectively.The viability of SH-SY5Y cells were measured by MTT,and Western Blot was used to detect the changes of LRRK2 related signal pathway.3.We employ Male flies were selected and randomly divided into five groups,and treated with different concentration of resveratrol and soy isoflavone,respectively.The living survival and behavior change of flies were measured and recorded.Western Blot was used to detect LRRK2 kinase activity and the expression of LRRK2 related signal pathway protein.Results:1.Obtained rotenone-reduced PD cells,GS2 transgenic flies,and transgenic mice with the genotype of GS2-GS2,GS2-A53T,GS2,A53T and non.2.Compared with control group,SH-SY5Y cells treated with medicine have a higher survival rate,and the optimum effect of resveratrol and soy isoflavone are all 100 nM.Western Blot results shows that resveratrol and soy isoflavone in different concentration have different activation effect on Nrf2/ARE and ERK,JNK,P38 signal pathway.3.Compared with control group,flies treated with medicine have longer living survival,better moving ability.In different medical concentration,100 ?M resveratrol and 1 ?M soy isoflavone have optimum effect.Western Blot results shows that resveratrol and 1 ?M soy isoflavone can inhibit LRRK2 kinase activity,and influence the signal pathway of Nrf2/ARE-oxidative stress related pathway,and MAPK family related ERK,JNK,P38 pathway.Conclusion:1.Successfully established rotenone-reduced PD cell model,GS2 transgenic fly model,generated homozygosis GS2 and GS2-A53T double transgenic mouse model.2.Resveratrol and soy isoflavone have evidently protection on PD related cell models,and the optimum concentration are 100 nM,the molecular mechanism of which can be the activation of Nrf2/ARE and JNK,ERK,p38 signal pathway.3.Resveratrol and soy isoflavone can obviously improve PD symptom in flies,inhibit LRRK2 kinase activity,and 100 ?M resveratrol and 1 ?M soy isoflavone works best.Resveratrol works on Nrf2/ARE and JNK,ERK pathway,and soy isoflavone works on p3 8,ERK pathway.