Mechanism Of Protective Effect Of Astragaloside IV Against Zinc Oxide Nanoparticles Induced Neurotoxicity

Objective:Zinc oxide nanoparticles(ZnONPs),as widespread nanoparticles materials,which exhibit wide application in biology,medicine,and food fields.The potential neurotoxicity effect of ZnONPs has raised concerns.Astragalus membranaceus is a Traditional Chinese Medicine that has the effects of invigorating qi for ascending Yang,invigorating qi and consolidation of superficies.Modern pharmacological research has also indicated that the active ingredients from astragalus have the potential effects in cardiotonic,ameliorating inflammatory and oxide stress,antiviral,immune regulation,regeneration and repair in Central Nervous System.Previous studies in our research group have demonstrated that ZnONPs can induce mitochondrial pathway-mediated apoptosis results in the SH-SY5 Y human neuroblastoma cell death.In this study,the mechanism of ZnONPs-induced neurotoxicity ameliorated by active components from astragalus membranaceus will be explored,which laid the foundation of the neuroprotective activity of Astragalus.Methods:1.The human neuroblastoma cell line SH-SY5 Y cells were used as a research subject.In absent and present of Astragalus,APS,ASIV and Calycosin,the cell viability of 50 nm ZnONPs on SH-SY5 Y cells was detected by CCK 8 and LDH assays.2.Apoptosis rate of ASIV against the neurotoxicity of ZnONPs at the low,medium and high doses was detected by Annexin V-FITC/PI double staining.Western blot was used to examine the apoptosis-related proteins expression of caspase 9,caspase 3,BCL-2,and Bax.3.The autophagy marker protein LC3 fluorescent density of ASIV against the ZnONPs-induced neurotoxicity was observed by Immunofluorescence.And the autophagy-related proteins level,such as p62,LC3Ⅱ,Beclin1,was evaluated by Western blot.The cell viability in the presence of autophagy inhibitor(3-MA)and autophagy agonist(rapamycin)was detected,respectively.4.Immunofluorescence was used to detect the co-location of mitochondrialautophagosomes-lysosomes and the protein expression of PINK1.And the protein level of PINK1,Cyto-Parkin,Mito-Parkin was examined by Western blot.Mitochondrial protective of ASIV against ZnONPs was examined that the mitochondrial membrane potential,mitochondrial mass,the fluorescent intensity of ROS and the intracellular calcium ion concentration were detected by JC-1 staining,NAO staining,flow cytometry,respectively.Results:1.ZnONPs could significantly decrease the SH-SY5 Y cell viability.Astragalus had a protective effect on ZnONPs-induced cytotoxicity.Astragaloside IV,one of the main active components of astragalus,had a protective effect on the ZnONPs-induced cytotoxicity.Astragalus polysaccharide and Calycosin had not.2.The ZnONPs-induced apoptosis in SH-SY5 Y cells was increased at the presence of ASIV at 12 h and 24 h,respectively.The expression of caspase 3,caspase 9,BCL-2 were down-regulated and Bax was up-regulated,which was dose-dependent and time-dependent.3.A significant decrease of p62 expression and increase of LC3Ⅱ and Beclin1 levels were observed with the exposures of ASIV and ZnONPs to SH-SY5 Y cells for 24 h.The expression and fluorescent intensity of Immunofluorescence of LC3 punctate were significantly increased.And the neuroprotective of ASIV was abolished by the interference of 3-MA and enhanced by RAPA.4.Increase fluorescent intensity and quantity of PINK1 were found by Immunofluorescence.We used Lyso-Tracker Green and Mito Tracker Red to characterize the Lysosomes and mitochondrial,respectively.The co-location of LC3-mitochondrial and mitochondrial-lysosomes were observed.The increase expression levels of Mito-Parkin and decrease of PINK1,Cyto-Parkin were observed.The results showed that the PINK1/Parkin mediated mitophagy was activated with the interaction of ASIV and ZnONPs.Moreover,the mitochondrial mass decrease,ROS released and MMP increases,calcium ion homeostasis disequilibrium caused by ZnONPs against SH-SY5 Y cells were reversed by ASIV.Conclusion:1.ASIV,an active component from Astragalus,could reduce the neurotoxicity of ZnONPs induced.2.The ZnONPs induced apoptosis was increased by ASIV.3.Activation of autophagy and PINK1/Parkin-mediated mitophagy caused by ZnONPs against SH-SY5 Y cells was increased by ASIV,which ameliorates the neurotoxicity and mitochondrial dysfunction.4.The ASIV exerted a protective effect on ZnONPs-induced neurotoxicity through activating autophagy/mitophagy of SH-SY5 Y cells.