Graphene Oxide Enhances β-amyloid Clearance By Inducing Autophagy Of Microglia And Neurons

Alzheimer’s disease(AD)is the highest proportion of dementia in the world andβ-amyloid(Aβ)occupies the leading position among its pathogenic factors.A large amount of Aβ aggregates to form plaques distributed around neurons in the brains of AD patients.On the one hand,Aβ directly damages neurons because of its own toxicity,and on the other hand,it indirectly damages neurons by affecting microglia to increase inflammation.The main reason for Aβ accumulation is that the Aβclearance ability is greatly reduced.Therefore,solving the obstacle of Aβ clearance is a key solution to protect neurons and treat AD.The clearance pathway of Aβ is not limited to the protease degradation and the efflux of the blood-brain barrier.The autophagy-lysosome system is also one of the main clearance pathways,and its dysfunction primarily affects the metabolic balance of Aβ.In the autophagylysosomal clearance system,the clearance of Aβ by microglia and neurons through the autophagy pathway is considered to be one of the most important safest ways to clear Aβ.However,both cells have autophagy dysfunction in the pathological environment of AD.Therefore,it is a safe and feasible way to restore the balance of Aβ metabolism by regulating the autophagy function of the two cells to promote the degradation and clearance of Aβ.Graphene oxide(GO),a graphene-derived nanomaterial,has high stability in water dispersion compared with the original graphene.Research has been done in the cultivation of induced pluripotent stem cells,cancer diagnosis,drug delivery,cell growth,etc.Studies have found that GO also has a superior effect on regulating autophagy in recent years,but there is no exploration,research and application of GO in the field of Aβ clearance.Therefore,it is necessary to explore whether GO can promote the clearance of Aβ by inducing autophagy of microglia and neurons to protect neurons and treat AD.To sum up,the primary research goals include: 1)To explore whether GO has the ability of inducing autophagy in microglia and neurons.2)To explore the pathway and mechanism of GO-induced autophagy.3)To explore whether GO can promote the clearance of Aβ by inducing autophagy in microglia and neurons.4)To explore whether GO can protect neurons.Methods:The large-size GO was subjected to ultrasound treatment for 36 hours and its size and shape were observed under the transmission electron microscope to obtain the small-size GO that meets the size requirements of crossing the blood-brain barrier.The experiment used BV2(microglia)cells and SH-SY5Y(neurons)cells.After treating the two kinds of cells with GO,the MTT and Western Blot(WB)methods were used to confirm whether GO is toxic to the two kinds of cells and whether GO can affect autophagy in two kinds of cells,so as to realize the conditions screening of GO.Then each type of cell was set to four groups: CON(PBS)group;GO50(50μg/ml GO)group;GO100(100 μg/ml GO)group;Rap(rapamycin)group according to experimental design.The WB and the acridine orange were used in the experiment to detect autophagic flux and stain the autophagic vesicles.After co-incubating BV2 cells with Aβ-FITC,the Aβ MFI in BV2 cells was measured.At the same time,BV2 cells and SH-SY5 Y cells were co-cultured and incubated with Aβ in order to simulate a more real AD pathological environment.The changes of LC3 and the MFI of Aβ in the two cells was detected to judge the degradation of Aβ under co-culture.In addition,the experiment also explored the protective effect of GO on neurons through real-time observation of neurons growth.When exploring GO-induced autophagyrelated pathways,we added compound C(AMPK specific inhibitor)and divided each type of cell into three groups: CON(PBS)group;GO100(100 μg/ml GO)group;GO100+compound C group.The changes of related pathway proteins that may be involved in the mechanism of GO-induced autophagy are detected by WB.Results:1.GO showed no cytotoxicity to BV2 cells and SH-SY5 Y cells.2.GO up-regulated autophagy in BV2 cells and SH-SY5 Y cells.3.GO promoted microglia-mediated phagocytosis of Aβ.4.GO accelerated the degradation of Aβ by improving the autophagy ability of BV2 cells and SH-SY5 Y cells under pathological conditions.5.GO protected the SH-SY5 Y cells under pathological conditions.6.AMPK and m TOR were involved in the process of GO up-regulating autophagy in BV2 cells and SH-SY5 Y cells.Conclusion:GO simultaneously activated autophagy of microglia and neurons through the AMPK/m TOR pathway,thereby promoting Aβ clearance and ultimately protecting neurons.The good ability of GO to activate autophagy and clear Aβ reflects its great potential in the treatment of AD.