| Objectives: Autophagy is a metabolic process of cell degradation of abnormal organelles and proteins,which plays an important role in maintaining the homeostasis of the intracellular environment.Neurons are special permanent cells in the human body,highly dependent on autophagy maintenance of cell homeostasis and are necessary for high levels of ATP and protein synthesis.Alzheimer's disease(AD)is a kind of neurodegenerative diseases.Autophagosome accumulation in axon terminal and subsequent autophagic dysfunction play key role in the onset and development of AD.Curcumin,as a polyphenol plant compound,has been shown to enhance autophagy in AD,but its exactly mechanism is not clear.In this study,the effects of curcumin on autophagosome were observed by AD cell model.Then the effects of Curcumin on autophagy axonal transport were observed.Further more,the role of Curcumin on autophagy axonal transport by regulating forward molecular motor and/or reverse molecular motor were analyzed.Finally,whether this effect is due to regulating molecular motor were discussed.This study discusses the mechanism of curcumin's protective effect on AD from a new perspective,which is expected to provide new ideas for the treatment of AD.Methods: The N2a/APP695 swe cells were constructed and the cells were divided into four groups: WT group(WT/N2 a cell group),APP group(N2a/APP695 swe cells),DMSO group(5 umol/l DMSO solution treated with N2a/APP695 swe cells for 24 h)and Curcumin(CUR Group(5 umol/l curcumin treated N2a/APP695 swe cells for 24 h).First of all,the electron microscope was used to observe the autophagosome structure,immunofluorescence was used to detect the expression of autophagy marker protein LC3 and P62,RT-PCR and Western Blot were used to detect the mRNA and protein expression levels of LC3 and P62,respectively.Western Blot was used to detect the expression of autophagy related proteins Beclin1,Atg5 and Atg16L1.Immunohistochemistry and Western Blot were used to detect the expression of lysosome-labeled protein LAMP2.Confocal microscopy was used to observe the association of autophagosomes with lysosomes.Finally,lysosomal inhibitors were used to further quantify autophagic flux.Western Blot was used to detect the expression of kinesin(KIF),Dynein(DIC,DHC and DLC)and dynein-related proteins P150,P50,BICD2 and scaffolding proteins Huntingtin and RILP.Confocal microscopy was use to observe the colocalization of LC3 and BICD2.Results: Compared with APP group,the number of autophagosomelike structures in Curcumin group and WT group were obviously increased,and the expression of LC3 and the level of LC3 mRNA were increased,but the expression of P62 and the level of P62 mRNA were decreased.The expression of LC3 II,Beclin1,Atg5 and Atg16L1 in Curcumin group were higher than that in APP group,but P62 expression was decreased in Curcumin group.Immunohistochemistry and immunofluorescence showed that the expression of LAMP2 and the number of autophagy lysosomes were increased in WT group and Curcumin group,and the expression of LC3 II and P62 were increased with barphramycin.After that,Curcumin could increase LC3 II slightly and decrease the expression of P62.The expression of DIC,DHC and DLC were increased,but the expression of KIF was decreased,and the expression of LC3 II and P150 was decreased after EHNA treatment.Curcumin could increase the expression of LC3 II induced by EHNA and decrease the expression level of P62.The expression of P150,P50,BICD2,RILP and Huntingtin were also increased in Curcumin group,and the co-localization of BICD2 and LC3 was observed in Curcumin group,and Curcumin promoted the formation of Dynein-Dynactin-BICD2 complex.Conclusion: Curcumin can induce the autophagy of N2a/APP695 swe cells,improve the fusion of lysosomes and autophagosomes,increase the expression of axon transporter-related proteins,enhance the axon transport of autophagic phages and finally promote autophagy flux,which provide a new theoretical basis for AD treatment by Curcumin. |
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