The Mechanisms Of Graphene Oxide And Reduced Graphene Oxide Induced PC12 Cells Apoptosis And Cell Cycle Alterations

BackgroundGraphene material is a new type of nano materials found in 2004,which have a good application prospect in the field of bio-materials.Graphene and its derivatives,such as graphene oxide(GO)and reduced graphene oxide(rGO),have been applied as bioimaging technology,medicine and gene delivery,tissue engineering,etc.In the oral application fields,using the graphene derivatives to modificate the implant and denture base can greatly improve their performance compared with the traditional materials.However,graphene and its derivatives can also enter into human body through various ways,during their research and development,production and application.After that,they enter into tissues,organs and cells,and produce toxic effects.Studies have shown that graphene nanomaterials can penetrate the blood brain barrier and enter into the brain,but the detailed mechanism on neurotoxicity is not clear yet.ObjectiveThis study used the PC 12 cells to simulate the neuronal cell model.Cytotoxicity evaluations were applied after GO or rGO treatment.In addition,toxic effects on the apoptosis and cell cycle change were detected at same dose to further analysis the nanotoxicology of GO and rGO.Materials and methodsChapter 1:Characterization and cytotoxicity evaluation of GO and rGO1.The original particle size and morphology of GO and rGO were detected by atomic force microscopy(AFM).The particle size and zeta potential of the nanoparticles in the suspension of water were measured by dynamic light scattering(DLS).The Raman spectra characterized the defects(D peak)of graphene materials,the in-plane vibration(G peak)of sp2 carbon atoms,and the stacking mode of carbon atom(G' peak).2.The neuronal like changes of PC 12 cells were induced by nerve growth factor(NGF)and the neuron model was established.After GO or rGO treatment,the survival rate of PC 12 cells were detected by CCK-8 toxicity-proliferation test,and the release of lactate dehydrogenase(LDH)from cells were detected by LDH release kit.Chapter 2:GO and rGO induced apoptosis and cell cycle changes of PC12 cells1.The effect of GO and rGO on the apoptosis and necrosis of PC 12 were detected by the apoptosis detection kit.2.The cell cycle alteration of GO and rGO on PC 12 were detected by cell cycle detection kit.Immunofluorescence was used to observe the change of cell division situation.Chapter 3:The study of GO and rGO induced PC12 cytotoxicity and the related mechanisms1.The ultrastructural changes of mitochondria in the cells were observed by transmission electron microscope,the changes of mitochondrial membrane potential and relative reactive oxygen species(ROS)generation in the cells were detected by flow cytometry,and the intracellular ATP were detected by Luminometer.2.The total RNA in the cells was extracted,and the expression of apoptosis and cytoskeleton related genes were detected by quantitative real-time PCR.3.The effects of GO and rGO on the cytoskeleton system of PC 12 were observed by immunofluorescence.4.The effects of GO and rGO on the cytoskeleton related protein and the level of phosphorylation of extracellular regulated protein kinases(ERK)signaling pathway in PC 12 were detected by Western-blot.Results1.AFM showed the GO and rGO used in this experiment were irregular flakes,both of their thickness were less than 1 nm;Raman spectroscopy detected the graphene oxide peaks:G peak,D peak,G' peak;DLS results showed that the average diameter of GO was 219 nm,the Zeta potential was-14.3+11.1 mV,rGO average particle size was 122.4 nm,the Zeta potential was-17.7+7.99 mV.2.The PC 12 neuron model was successfully induced.GO and rGO exhibited different cytotoxicity under concentration gradients,and induced intracellular LDH release.3.Higher concentration of GO and rGO could induce PC 12 apoptosis and necrosis,they could also induced PC 12 cell cycle mainly arrest at G0/G1 stage and inhibited cell division.4.GO and rGO induced cellular mitochondrial function damage and the expression levels changes of apoptosis related genes in cells.5.GO and rGO damaged the cytoskeleton system in cells,and induced expression levels changes of cytoskeleton related genes and proteins in cells.6.GO and rGO regulated the phosphorylation level of ERK signaling pathway.ConclusionGO and rGO can induce PC 12 cytotoxicity,apoptosis and necrosis,and inhibited the normal cell cycle and cell division.Mitochondrial dysfunction induced by GO and rGO mediates apoptosis and necrosis.GO-and rGO-induced microtubule and actin filaments damage are the major reasons in regulating cell cycle and mitotic changes.Moreover,the phosphorylation level alterations of ERK pathway after GO and rGO treatment play an important role in mediating apoptosis and cell-cycle arrest.