Galantamine Inhibits β-amyloid-induced Cytostatic Autophagy In PC12 Cells Through Decreasing ROS Production

Objectives Alzheimer’s disease(Alzheimer disease AD)is one of the most common neurodegenerative disease,and is one of the most prevalent brain diseases among the elderly,majority of which is caused by abnormal deposition of amyloid beta-peptide(Aβ),tau protein phosphorylation level agglomeration in neurons of neural neurofibrillary tangles(neurofibrillary tangles,NFT)and loss of neurons and synaptic changes.Galantamine,currently the first-line drug in treatment of AD,has been shown to diminish Aβ-induced neurotoxicity and exert favourable neuroprotective effects,but the detail mechanisms remain unclear.This study will elucidate the molecular mechanism of galanthamine inhibits Aβ-induced autophagy in PC12 cells,to provide theory evidence and experimental basis for the further analysis of the pathogenesis and the clinical treatment of AD.Methods(1)To explore the influences of galantamine on Aβ-induced cytotoxicity in PC12 cells by MTT and clone formation method.(2)To explore the effects of galanthamine on Aβ-induced autophagy in PC12 cells by Western blotting and immunofluorescence experiments.(3)To analyze the effects of galanthamine on Aβ-induced apoptosis in PC12 cells by TUNEL and Hoechst staining and Western blotting assay.(4)Western blotting and dichloro-dihydro-fluorescein diacetate(DCFH-DA)assay were used to explore how galanthamine affects the expression of NOX4 and ROS produced and inhibits Aβ-induced cytostatic autophagy in PC12 cells.(5)Immunohistochemical technique was used to analyze the effect of galanthamine on neuron injury mediated by Aβ in animal level.Results(1)Galantamine reversed the Aβ-induced cytotoxic effect in PC12 cells.The results of MTT experiment shown that treatment of Aβ for 24 hours suppressed the cell viability in a dose-dependent manner,where IC50 was approximately 2 μmol/L.Galantamine treatment after 24 h effectively reduced the toxicity of PC12 cells induced by Aβ,and the effect was most obvious in the treatment of the addition of 40 μmol/L.In addition,this protective effect was further supported by clone formation assay,in which treatment of galantamine significantly reversed the proliferation of Aβ-treated PC12 cells.(2)Galantamine inhibited the autophagy induced by Aβ in PC12 cells.The results of Western blot and immunofluorescence shown that treatment of PC12 cells with Aβ was leaded to the formation of autophagy flow and autophagosomes accumulation,and galanthamine effectively inhibited the autophagy of PC12 cells induced by Aβ.(3)Galantamine inhibited the apoptosis induced by Aβ in PC12 cells.Detected the apoptotic rate through TUNEL assay.As expected,Aβ induced significant apoptosis in PC12 cells,which can be partly reversed by the treatment of galantamine.Furthermore,nuclei were stained with Hoechst 33342 to evaluate the apoptotic level.As the results shown,the nuclei exhibited highly concentrated and fragmented morphology,and co-treatment of 40 μmol/L galantamine reduced the number of condensed and apoptotic cells.Additionally,we also determined the expression of cleaved caspase-3,and the results were consistent with above TUNEL assay and Hoechst staining.The results suggested that galantamine inhibits Aβ-induced cytostatic autophagy in PC12 cells.(4)Galantamine inhibits Aβ-induced autophagy through inhibiting the expression of NOX4 and decreasing ROS accumulation.DCFH-DA fluorescence probe and Western blot were used to measure the production of ROS and expression of NOX4,the data showed that Aβ dramatically increased the ROS accumulation and expression of NOX4 in PC12 cells,which was mostly reversed by the treatment of galantamine.LPS abolished the galantamine-induced autophagy suppression when the addition of LPS incubated cells.ROS scavenger NAC was used as positive control,the effect of NAC is the same as galanthamine.The results showed that the galanthamine inhibits Aβ-induced autophagy in PC12 cells through suppressing the expression of NOX4 and production of ROS.(5)The animal experiment evaluated galanthamine to protect the neurons by inhibiting the autophagy mediated by Aβ.The changes of autophagy related protein markers(LC3,Beclin-1,etc.)and neuron damage marker neuron-specific enolase(NSE)in hippocampal tissue of rat cerebral cortex were detected by immunohistochemistry.The results showed that galanthamine can effectively inhibit the expression of LC3 and Beclin-1 mediated by Aβ,and galanthamine also inhibited neuron injury mediated by Aβ,galanthamine inhibited the expression of NSE mediated by Aβ.The results show that galantamine to protect the neurons by inhibiting the autophagy mediated by Aβ.Conclusion Galantamine inhibits Aβ-induced cytostatic autophagy through inhibiting the expression of NOX4 and decreasing ROS accumulation,it has a neuroprotective effect on the neuron.Providing new insights and mentality into deep understanding of AD progression and molecular basis of galantamine in neuroprotection.