Effect Of Abscisic Acid On αA53T Induced Parkinson’s Disease Model

Abscisic Acid(ABA)is an important hormone in plants;while it is also associated with some diseases in animals,inflammatory reactions and so on.Clarifying the binding receptors of abscisic acid in mouse brain: Lanthionine synthase C-like protein,Peroxisome Proliferator Activated Receptor and their distribution in brain regions;exploring the effect of ABA on Parkinson’s disease phagocytosis and apoptosis markers.ObjectiveIn this study,by constructing mouse model of Parkinson’s disease,we will clarify the binding receptors of ABA in the mouse brain: LANCL2,PPARγ and their distribution in brain regions.The constructed α A53 T over expressing mn9 d Parkinson’s disease cell model was intervened with ABA to explore the changes of apoptosis and related autophagy markers in cells in the Parkinson’s disease cell model with different μM ABA.Methods(1)Twelve C57BL/6J male mice were randomly divided into two groups: Saline and Parkinson’s disease model(MPTP).Behavioral test analysis was performed on mice after the model was established 7 days,and brain regions were isolated by perfusion and then cryopreserved.The distribution of LANCL2 and PPARγ in different brain regions of mice was tested by immune histochemical staining.(2)The recombinant plasmids were amplified and verified,and the results of DNA agarose gel electrophoresis and the company’s sequencing showed that the recombinant plasmids were amplified correctly.Wild type mn9 d cells were transiently transfected to construct a Parkinson’s cell model overexpressing α WT and α A53 T.(3)The contents of LANCL2 and PPARγ in wild cells were observed and detected.Western Blot detected the expression of α syn,LC3,Caspase-3,LANCL2 and PPARγ.(4)The mn9 d cells were intervented with different levels of Abscisic Acid.The expression of autophagy and apoptosis marker proteins in cells of overexpressing α WT and α A53 T mn9d after Abscisic Acid intervention were detected by Western Blot and Immuno Fluorescence assays.Results(1)In wild type mice,the expression of LANCL2 was higher in the striatum,hippocampus and substantia nigra,and the expression of PPARγ was higher in OB.The MPTP drug treatment group was relatively less.(2)DNA agarose gel electrophoresis found α WT and α A53 T bright bands appeared near the 400 bp marker,and then the genes were transfected into mn9 d cells.The protein content of α syn was compared in mn9 d cells transfected with the target gene: α A53 T > α WT > vector.Immuno Fluorescence experiments showed that α syn protein aggregated,and LC3 also appeared brighter spots.Observing and comparing the fluorescence density of apoptosis marker Caspase-3 found that the pathological apoptosis of cells increased.In conclusion,the PD cell model was successfully constructed.(3)α syn and LANCL2,PPARγ proteins were positively correlated in mn9 d cells transfected with αsyn WT and α syn A53 T.(4)The comparison of the mean gray value of autophagy protein LC3 in mn9 d cells treated with different μM ABA showed that α syn A53T(0)>α syn WT >50 >100 >200.Compared with PD group,200 μM ABA group had a significant difference,P<0.05.IF observation results: αA53T(0)>50 >200 >100,mean fluorescence density: Compared with the PD model group,the experimental group had a very significant difference in 200 μM ABA,p<0.01.The LC3II/LC3 I ratio decreased,indicating a decrease in autophagy.In conclusion,ABA reduced the occurrence of autophagy,and the PD model group was significantly different from the experimental group with 200 μM ABA,P<0.05.(5)After adding exogenous ABA,the BAX protein content decreased,but the Bcl-2 protein content increased.The Bcl-2/BAX value increased,indicating that apoptosis was reduced.The effect of different content(μM)ABA on reducing apoptosis was: 200 >100 >50 >0.Compared with the PD group,the 50 μM and 100 μM groups had significant differences,P<0.05,and the 200 μM groups had extremely significant differences,P< 0.01.Conclusions(1)Nuclei colocalization analysis showed that most of PPARγ was localized in the nuclei of the OB brain region;LANCL2 was mainly localized in the nuclei of the SN brain region of mice.(2)There was no significant difference in the expression of ABA receptors LANCL2 and PPARγ in wild type mn9 d cells.(3)α syn and LANCL2,PPARγ were positively correlated in mn9 d cells transfected with α syn WT and α syn A53 T.(4)The expression of Beclin1 decreased,the value of LC3II/LC3 I decreased,and Bcl-2/BAX increased.It can be seen that the autophagy of cells transfected with α A53 T gene decreased after ABA intervention.(5)In the current trial,ABA was found to reduce the effects of pathological autophagy and apoptosis in Parkinson’s disease cells.