| ObjectiveAlzheimer’s disease(AD),a common neurodegenerative disease in the elderly,the exact cause of the disease has not been clear,up to now β-amyloid deposition to form senile plaques is still its relatively mainstream pathogenesis.In recent years,lysosomal dysfunction has made cellular waste accumulation lead to the pathogenesis and development of AD.This pathological understanding has provided a new research direction for the prevention and treatment of AD,and promoting cellular waste removal by enhancing lysosomal activity has become an attractive research direction.Gemfibrozil(Gem)can promote TFEB nuclear translocation by activating the transcription factor PPARα,and then promote the biogenesis of lysosomes,and enhance the activity of proteolytic enzymes in the lysosomes.However,due to its poor water solubility and difficult to cross the blood-brain barrier,it greatly limits its clinical application.Exosomes(Exo)have lipid bimolecular structure,which provides the possibility of Exo effective drug loading,which can enhance the solubility,stability and bioavailability,and can be targeted by its modification.This study intends to use modified macrophage-derived exosomes to load gemfibrozil to discuss its research on mechanistic aspects of ameliorating cognitive impairment in AD mice.MethodsThis study first established the UV spectrophotometry(UV)for Gem content detection,And the linearity,precision,recovery rate and other methods are verified;MExo-Gem was prepared by gradient ultracentrirocentrifugation and incubation,Polyacrylamide gel electrophoresis and thin-layer chromatography were used to investigate whether the exosomes were successfully targeted and modified,Transmission electron microscopy(TEM),particle size analyzer,protein immunoblot(Western Blot)were used to investigate the morphology,particle size and marker protein of MExo-Gem;Its ability to accumulate and target in the brain to microglia was confirmed by in vitro and in vivo immunofluorescence experiments.In vitro experiments,the mechanism of MExo-Gem in the cell model stimulated with Aβ1-42 was explored by Western Blot,immunofluorescence,and Elisa experiments.Meanwhile,in in vitro experiments,the improvement of cognitive impairment in AD model mice was investigated by water maze method.Further validation of the mechanism of MExo-Gem in a mouse model of AD was performed by Nissl staining and immunofluorescence staining.ResultsThe methodological parameters in Gem’s UV quantitative analysis method all meet the methodological requirements.The results of polypropylene gel electrophoresis and TLC experiments indicated successful exosome targeted modification.A series of exosome characterization experiments confirmed that MExo-Gem structure was complete and particle size,and drug loading and modification had no effect on its structure and properties.The in vitro and in vivo cell uptake experiment results showed that MExo-Gem can accumulate in the brain and significantly colocalize with microglia.The experimental results of Western Blot method,immunofluorescence method and ELISA can show that MExo-Gem can promote TFEB nuclear translocation by activating PPARα,and then promote the biogenesis of lysosome and reduce the accumulation of Aβ in cells and brain.The results of water maze experiment,immunofluorescence experiment and Nissl staining showed that MExo-Gem could significantly reduce Aβ1-42 accumulation in mouse brain,reverse neuronal damage,and then significantly improve cognitive impairment in AD model mice.ConclusionsOur results show that mannose-modified exosomes loaded with Gem(MExo-Gem)can accumulate in the hippocampus through the blood-brain barrier,and specifically target the interaction between the microglia and mannose receptors expressed in microglia,and increase the concentration of drugs in microglia.MExo-Gem enters intracellular drug release to enhance lysosomal activity and further promote Aβ clearance in MG to reduce the total Aβ in the brain and improve learning and memory in AD model mice. |
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