Neuroprotective Effects Of Soy Isoflavones In LRRK2^G2019S And ?-Syn^A53T Double Transgenic PD Animal Models

Parkinson's disease(PD)is an age-related neurodegenerative disease.It is estimated that nearly 100,000 people in China become new patients with Parkinson's disease each year.The pathological features of PD include the appearance of characteristic eosinophilic inclusion bodies in the cytoplasm of neurons,the Levy body,and the loss of dopaminergic(DA)neurons in the nigrostriatal system.Clinical manifestations include resting tremor,myotonia,bradykinesia,and abnormal posture gait.In order to better understand the pathogenesis of PD and its potential therapeutic targets,this experiment mimicked PD drosophlia and mouse models of mutant LRRK2 G2019S and ?-synuclein A53T,and screened a better double transgenic animal model of PD.A lifetime intervention was given to the constructed PD Drosophila model with 1?M soy isoflavone.18-month-old PD mice were gavaged(ig)at 30 mg/kg for 14 days.The results showed that soy isoflavones can improve the lifespan and the behavior of PD fruit flies.The expression levels of TH,Nrf2 and GCLC were increased in the striatum and striatum,and the expression levels of ?-synuclein,p-LRRK2,p-JNK MAPK and p-p38 MAPK were decreased,and the dopamine transporters DAT and VMAT2 were not significantly increased.These results suggest that soy isoflavones may effectively inhibit LRRK2 kinase activity and reduce the protein expression level of ?-synuclein through the Keap1-Nrf2-ARE pathway and MAPK pathway,thereby achieving neuroprotective effects against Parkinson's disease.Objective:To establish a PD animal model.A certain concentration of soy isoflavones was administered to observe the effects of drugs on the morphology and function of the Drosophila model and the mouse model.Furthermore,the mechanism of action of soy isoflavones to improve PD was explored.Methods:1.Firstly,the A53T/G2019S double transgenic homozygous Drosophila was obtained by gene isolation and recombination.Then,the transgenic PD Drosophila model was established by UAS-GAL4 system,and the LRRK2 G2019S and ?-Syn A53T were localized in dopamine neurons and whole neurons.The Elav series:SynA53T/Elav;,Elav/+;LRRK2G2019S/+,SynA53T/Elav;LRRK2GS2/+,TH series:SynA53T/+;TH/+,;LRRK2G2019S/TH,SynA53T/+;LRRK2GS2/TH.their life cycle and athletic ability should be detected.2.The mouse model of LRRK2 transgenic PD was established,and the heterozygous mice expressing the LRRK2-G2019S were crossed with each other.The homozygous GS2 mouse model was obtained by PCR and cross-section identification.In addition,GS2 heterozygous mice were crossed with A53T heterozygous mice to obtain GS2-A53T double transgenic mice.Open field,rotarod,and climbing pole were used to evaluate the motility of WT,Syn-A53T(HET),LRRK2-G2019S(HOMO),LRRK2-G2019S0(HET),and GS2-A53T double transgenic mouse models.the expression levels of PD-related proteins in five model mice were examined by western blotting.3 Identify A53T/GS2 fruit flies and mice as our research subjects.Female flies were picked and divided into three groups.The model flies were fed with 1 um of soy isoflavones,and their life cycle and behavioral changes were observed.Mice were given 30 mg/kg(i.g.)to detect the changes in behavior before and after the administration.4.Immunohistochemistry was used to detect the expression of TH in the substantia nigra and striatum of mice,and the expression of a-synuclein in the substantia nigra pars compacta was detected by immunofluorescence.The expression of related proteins with PD-associated proteins,protein levels for DA transport,Keapl-N rf2-ARE pathway and MAPK pathway was also examined.Results:1.Successfully obtained Drosophila Elav series:SynA53T/Elav;,Elav/+;LRRK2G2019S/+,SynA53T/Elav;LRRK2GS2/+,TH series:SynA53T/+;TH/+,;LRRK2G2019S/TH,SynA53T/+;LRRK2GS2/TH.And in their life cycle tests,it was found that the life cycle of the three modes of Drosophila was shortened compared with the control group;LRRK2G2019S/TH,SynA53T/+;LRRK2GS2/TH strains were significantly different,extracted Half of the life cycle was counted,and the lifespan of the double transgenic Drosophila significantly shortened.SynA53T/Elav;LRRK2GS2/+showed no significant difference in life cycle and exercise capacity compared with control fruit flies2.WT,Syn-A53T(HET),LRRK2-G2019S(HOMO),LRRK2-G2019S0(HET),GS2-A53T transgenic mouse models were successfully constructed,and their 18-month survival rate was statistically analyzed.The A53T/G2019S double transgenic mice showed relative premature death with other groups.On the one hand,the results of exercise performance evaluation showed that A53T(HET)showed no difference in exercise speed and cross-grid frequency compared with the control group.Compared with the control,the G2019(HOMO)group's exercise ability decreased in the speed of movement,across the central area,and in the detection of the rotating rod,and showed significant differences.In the climbing rod experiment,each group was prolonged in time-consuming compared with the control,but there was no significant difference.The A53T/G2019S group showed a significant decrease in the speed of movement and cross-grid as well as in the bat group and the A53T HET group.In addition,TH content was lowest in the A53T/G2019S group in the substantia nigra and striatum.The results showed that P-LRRK2 protein content was highest in G2019S HOMO and A53T/G2019S in striatum and substantia nigra.3.1?M soy isoflavones in the double transgenic Drosophila model can prolong the life cycle of Drosophila,and also improve the abilities of A53T;GS2 Drosophila,and analyze the changes in the ability of Drosophila during the fifth week.In the analysis of the exercise ability of the rats,the walking speed administration group of the double transgenic mice was significantly higher than that of the model group,and the statistical administration group across the central number of cells also showed an increasing trend.In the rotating rod experiment,the double transgenic mice had an increasing tendency at the rod time compared with the model group after the function of SI.In the climbing rod experiment,GS2;A53T mice had no difference in the time spent on climbing and the WT group and the model group by the SI.4,immunohistochemistry results showed that compared with GS2;A53T group,GS2;A53T group under the action of SI,GS2;A53T group SNpc-DA has a significant rebound trend;in the striatum,after drug action,TH The expression also has a pick-up trend,but there is no significant difference.The results of immunofluorescence showed that the GS2;A53T mice had a large amount of syuclein protein expression in the substantia nigra pars compacta of GS2;A53T mice.The expression of syuclein protein in the substantia nigra of the soybean isoflavone group was significantly lower than that of GS2;the A53T group significantly decreased.5.After the action of soybean isoflavones,the content of TH in the model group was significantly increased,the phosphorylation of LRRK2 was significantly decreased,and the DAT protein level did not change significantly after administration.The VMAT2 protein level could not be significantly improved after treatment.After the action of soybean isoflavones,the content of TH in the model group was significantly increased,the degree of phosphorylation of LRRK2 was significantly decreased,and the level of DAT protein did not change significantly after administration.The level of VMAT2 protein could not be significantly improved after treatment.The reduction of Nrf2 and GCLC caused by the A53T/G2019S mutation was preferably inhibited.When the MAPK pathway was explored,the degree of JNK phosphorylation was significantly increased in the JNK signaling pathway by 1?M soy isoflavones,and JNK phosphorylated under the action of soy isoflavones was significantly lower than that of double transgenic mice.This indicates that double transgenic mice may play a role in the JNK signaling apoptotic pathway through LRRK and synuclein.In the p38 signaling pathway,the degree of phosphorylation of p38 was significantly increased in the double transgenic mice compared with the control group,and phosphorylation under the action of 1?M soy isoflavones showed a downward trend but no significant difference.Conclusion:1.Successfully established A53T/G2019S double transgenic homozygous flies,which were crossed with Elav-Gal4 and TH-GAL4,respectively,to obtain specific mutations in specific parts.A GS2/A53T double-transgenic heterozygous mouse model,a GS2 homozygous transgenic mouse,and A53T/G2019S were identified as our PD animal model.2.Soy isoflavones significantly improved the exercise capacity and survival rate of double transgenic Drosophila,and improved the exercise ability of PD transgenic mice.3.The experimental results show that soy isoflavones improve the deletion of dopamine neurons and have no significant effect on the transport of dopamine.It may inhibit the increase and inhibition of Nrf2 and GCLC in Keapl-Nrf2-ARE pathway by inhibiting A53T/G2019S mutation.-JNK expression level;on the one hand.the expression of syuclein protein is attenuated,and on the other hand,LRRK2 kinase activity is inhibited,thereby slowing the pathological process of Parkinson's disease.