Neuroprotective Mechanism And Effects Of EGCG On LRRK2-G2019S Mutant Of Parkinson’s Disease

Objective:Leucine rich repeat kinase 2(LRRK2)is an autosomal dominant PD gene,and its genetic mutation is the most common.Statistics show that the family of genetic PD patients accounted for 10%of PD cases,LRRK2 mutations in the family of genetic PD patients accounted for about 7%of PD cases.Increased LRRK2 kinase activity caused by hereditary mutations is currently recognized as one of the pathogenesis of PD and supports the development and application of modern clinical drugs.Epigallocatechin-3-gallate(EGCG)is an active ingredient in Chinese herbal preparations and has been shown to be effective in inhibiting LRRK2 kinase activity in in vitro cytology experiments.In this experiment,the corresponding transgenic Drosophila strains were used to explore the mechanism of action of EGCG,to verify its inhibitory effect on kinase activity in vivo,and to screen out the optimal concentration and time.Further experiments were conducted to study the production of LRRK2 mutation.The mechanism of action of hereditary PD.Methods:Ddc-LRRK2-G2019S strains of Drosophila and control fruit flies were reared using standard corn culture medium.Drosophila were fed with GW5074,NAC and different concentrations of EGCG in medicated medium to detect and count the life cycle of Drosophila.And exercise ability,screening the optimal concentration and optimal time of drug action;Western blotting was used to detect the expression of p-LRRK2,p-p38 MAPK,Nrf2,p-ERK1/2 and TH in Drosophila brain tissue homogenate.1.Ddc-LRRK2-G2019S strains of Drosophila were given EGCG with concentration gradient,and control flies were reared using standard com medium.Drosophila of each experimental group were bred with GW5074,NAC and medicinal medium prepared with different concentrations of EGCG.Detect life cycle and exercise capacity and statistics,screen the optimal drug concentration,and use it for the next experiment during the period of significant changes in life cycle and exercise capacity.2.Ddc-LRRK2-G2019S strain Drosophila(or Ddc-GS2 for short)was used as the experimental group,and Ddc-Gal4 strain Drosophila was used as the negative control group,N-acetyl-L-cysteine and GW5074 was used as a positive control.After feeding for a certain period of time,the brain of Drosophila was taken out under a stereo microscope for observation,or the brain tissue homogenate was extracted and the p-LRRK2,p-p38 MAPK,Nrf2,p-ERK1/2 in Drosophila brain protein were detected by Western blotting.,TH expression.Determining the appropriate concentration of EGCG to treat PD due to LRRK2-G2019S mutations,prolonging the lifespan of PD model Drosophila and improving the possible mechanisms of PD model Drosophila motility.Results:1.Life cycle:EGCG significantly prolonged the lifespan of Drosophila and improved the ability of Drosophila to move.The life extension of EGCG group with the concentration of 10 μmol/L was the most obvious.And the life cycle curve changed significantly around 35 days.The statistical results of half survival time showed that the EGCG group with the concentration of 10 μmol/L had the most significant significance.2.Exercise capacity:The exercise capacity(climbing)of the EGCG group was the most obvious when the concentration was 10 μmol/L and 1 μmol/L.The statistics were meaningful in the fourth,fifth,sixth and seventh weeks.In the fifth week,the statistical significance was most obvious.3.Protein analysis showed that EGCG could increase the expression of Nrf2,p-ERK1/2 and TH in Drosophila brain and decrease the expression of p-LRRK2 and p-p38 MAPK.These results suggest that EGCG can effectively inhibit LRRK2 kinase activity,and the mechanism may be related to Keapl-Nrf2-ARE pathway and MAPK pathway.Conclusion:1.The effect of EGCG at a concentration of 10 μmol/L was relatively obvious,as evidenced by an improvement in life cycle and exercise capacity of around 35 days,that is,a major change at the fifth week.2.Possible signaling pathways interacting with EGCG:by activating Nrf2,which in turn increases glutamate cysteine ligase catalytic subunit(GCLC)gene expression,increases GSH expression,produces antioxidant effects,and increases cellular resistance to oxidative stress It protects dopamine neurons and has a certain therapeutic effect on Parkinson’s disease.EGCG can effectively inhibit LRRK2 kinase activity,possibly through p38 MAPK,p-ERK MAPK signaling pathway,to protect dopaminergic neurons from mutant LRRK2-induced neurotoxicity.