Study On The Mechanism Of Chinese Medicine Monomer Valeriaenoic Acid-co-loaded DCas9 VP64 MRNA And Its Sg RNA Nanoparticles On Alzheimer’s Disease

Objective:In this study,valeritic acid（Valerenicacid,VA）was used to treat dementia（Alzheimer’s disease,AD）,and poly（ethylene glycol）-polylactic acid（PLA）co-loaded valerienoic acid nanoparticles（MPEG-PLA-SS-VA NPs）were prepared.To observe the effects of MPEG-PLA-SS-VA NPs on learning and memory function,brain Aβdeposition,oxidative stress and brain glucose metabolism damage in APP/PS1 mice,and to explore the therapeutic targets in the early stage of AD.It can provide experimental basis for early treatment of AD with MPEG-PLA-SS-VA NPs,and upregulate GLUT1 with dCas9 VP64mRNA and its sg RNA technique to explore the possible therapeutic mechanism,in order to provide good service for clinical treatment of dementia.Methods:1.The modified solvent evaporation method was used to prepare MPEG-PLA-SS-VA NPs.The morphology,composition,particle size and drug loading performance of the nanoparticles were characterized and tested.2.The effects of MPEG-PLA-SS-VA NPs on the learning and memory function of APP/PS1 mice were recorded by new object recognition experiment and Morris water maze test.3.The effect of MPEG-PLA-SS-VA NPs on Aβexpression in the brain of APP/PS1mice was observed by measuring the level and expression of Aβin the brain of AD mice.4.The levels of inflammatory factors,the expression of microglial cell marker Iba-1and astrocyte marker GFAP,oxidative stress index and apoptosis in the brain of APP/PS1mice were detected.The effects of MPEG-PLA-SS-VA NPs on cerebral inflammatory response and oxidative stress were observed.5.Real-time PCR method was used to detect the expression of GLUT1 mRNA,LRP1mRNA,NF-κB mRNA gene in brain tissue.The changes of GLUT1,LRP1 and NF-κB protein in hippocampus were detected by Western blot method,and the effect of MPEG-PLA-SS-VA NPs on GLUT1,LRP1,NF-κB of glucose transporter pathway in brain was observed.6.The GLUT1 transporter was up-regulated by dCas9 VP64 mRNA and its sgRNA technology in vitro,and the effect of GLUT1 up-regulation on the expression of cells in vitro was observed.Results:1.The particle size,drug loading,entrapment efficiency and drug-loading performance of the nanoparticles were characterized and tested by the preparation of MPEG-PLA-SS-VA NPs.The results showed that the drug loading of MPEG-PLA-SS-VA NPs was 8.6%,the particle size was 716.65 nm,the potential was 10.42 MV,the release was 6%in 2 hours,27%in 1 day,76%in 3 days,86%in 6 days.Therefore,the result of 11^thdetermination was used as a follow-up experiment.2.To observe the effect of MPEG-PLA-SS-VA NPs on learning and memory function in APP/PS1 mice.The results showed that 1 h after the experiment training,the PI of VA and MPEG-PLA-SS-VA NPs groups to APP/PS1 new things increased,and the PI of MPEG-PLA-SS-VA NPs group to new things increased significantly.It is suggested that MPEG-PLA-SS-VA NPs can prevent short-term memory impairment in APP/PS1 mice.MWM test showed that VA group and MPEG-PLA-SS-VA NPs group can significantly shorten the escape latent period,swimming distance and path length of APP/PS1 mice in the original safety platform,increase the time of the rest platform quadrant and cross platform quadrant,especially MPEG-PLA-SS-VA NPs.3.To observe the effect of MPEG-PLA-SS-VA NPs on the changes of Aβin the brain of APP/PS1 mice.The results showed that soluble and insoluble Aβ_1-40-40 and Aβ_1-42-42 in VA and MPEG-PLA-SS-VA NPs groups were lower than those in APP/PS1 model group,especially in MPEG-PLA-SS-VA NPs group.Immunofluorescence showed that only a small amount of Aβwas produced in WT group,but the expression of Aβin APP/PS1 group and APP/PS1Blank NPs group was higher than that WT group.After treatment,VA and MPEG-PLA-SS-VA NPs could significantly decrease the expression of Aβ,especially MPEG-PLA-SS-VA NPs.4.The effects of MPEG-PLA-SS-VA NPs on brain inflammatory response,oxidative stress and apoptosis in AD mice were observed.The results showed that after treatment with VA and MPEG-PLA-SS-VA NPs,the contents of IL-1βand TNF-αdecreased significantly,IL-4 and IL-10 increased significantly,and the number of GFAP and Iba-1 positive cells decreased.Decreased staining intensity of MPEG-PLA-SS-VA NPs therapy can bi-regulate the protective effect of inflammatory factors on APP/PS1 mice.The results of oxidative stress showed that VA group and MPEG-PLA-SS-VA NPs group could significantly decrease the content of MDA,increase the levels of CAT and GSH-Px in brain tissue,increase the activity of SOD and protect nerve cells from oxidative stress.It may play a neuroprotective role by regulating the related indexes of oxidative stress.The results of apoptosis showed that the number of apoptosis in VA group and MPEG-PLA-SS-VA NPs group was lower than that in model group,especially in MPEG-PLA-SS-VA NPs group.5.To observe the effect of MPEG-PLA-SS-VA NPs on GLUT1,LRP1 and NF-κB in brain glucose transporter pathway.The results showed that compared with WT group,the amount of GLUT1 mRNA,LRP1 mRNA gene in hippocampus of model mice decreased significantly,and the quantity of NF-kB mRNA gene increased significantly.At the same time,WB further confirmed that the expression of GLUT1,LRP1 protein was significantly decreased,and the expression of NF-κB protein was significantly increased.Compared with the model group,the quantity of GLUT1 mRNA,LRP1 mRNA gene in hippocampus of VA group and MPEG-PLA-SS-VA NPs group was significantly increased,and the quantity of NF-κB mRNA gene was significantly decreased.Meanwhile,the expression of GLUT1 and LRP1 protein in hippocampus were significantly increased and the expression of NF-κB protein was significantly decreased in hippocampus of VA group and MPEG-PLA-SS-VA NPs group.Among them,MPEG-PLA-SS-VA NPS has a significant effect after treatment.6.The co-expression vector of sgGLUT1/dCas9VP64 was constructed by up-regulating GLUT1,by CRISPR dCas9VP64 and its sgRNA in vitro,and the effect of up-regulation of GLUT1 on N2A cells was observed.The results show that up-regulation of GLUT1 by 7kinds of sgRNA is not obvious,which may be related to the limited ability of activation domain selection and single sgRNA regulation.Conclusion:MPEG-PLA-SS-VA NPs can significantly improve the learning and memory function of APP/PS1 mice,decrease the deposition of Aβin the brain of APP/PS1mice,regulate the inflammatory reaction and oxidative stress,and inhibit cell apoptosis.The specific mechanism may be through regulating the change of glucose transporter pathway GLUT1/LRP1/NF-κB in the brain.