Effect Of Carbon Nanoparticles On Potassium Uptake And Oxidative Stress In BY-2 Cells

Carbon nanomaterials can boost plant biomass and upregulate the gene expression so as to stimulate plant growth.During the growth of plant,potassium is one of the main essentlial elements,which affects the yield and quality of potassium-loving plants.Carbon nanoparticles(CNPs)can promote the growth of plant and increase in the potassium content,but the mechanisms have not been discussed in depth.Therefore,in this thesis,CNPs were prepared by electrolysis of graphite,and then the mechanisms of promoting potassium uptake and the effect of oxidative stress by CNPs in BY-2 cells were studied,providing theoretical basis for stimulating plant growth and guidance to promoting growth of agricultural production.The main results are as follows:Firstly,the CNPs were prepared by electrolytic graphite,with good dispersity in water.The optimum condition for CNPs preparation was 0.1%ethylene glycol with current of 0.2A.CNPs exhibited particulate morphology and the size distribution was in the range from 18 to 70 nm,with an average size of 30 nm.Functional groups of COOH and OH were present on the surface of CNPs,and CNPs showed the common feature of graphitic sp2 hybridization carbons.The COOH groups on the surface of CNPs were functionalized via covalently binding with the Texas Red hydrazide for synthesis of the fluorescent carbon nanoparticles(FCNPs).The FCNPs were found to cross the cell membrane and enter cells in an energy-dependent manner and clathrin-independent endocytosis also mediated in the internalization of FCNPs in BY-2 cells.The K+accumulation increased with the increasing concentration of CNPs in BY-2 cellsand the potassium accumulation had a significant correlation with the fresh biomass of BY-2 cells.It was found that the mean value of K+influx reached 3797.50±942.46 pmol·cm2·s-1 after treatment with CNPs(concentration of 62.50 μg/mL).In the presence of potassium channel blocker TEA+,the K+accumulation was reduced significantly in cells.While the K+accumulation was not changed in the presence of CNPs inhibitors.In the presence of potassium channel blocker TEA+or CNPs inhibitors,the NKT1 gene expression was changed compared with the control group.These results indicated that CNPs upregulated potassium gene expression to enhance K+accumulation in BY-2 cells.The CNPs are selective for potassium ions.Moreover,CNPs were adsorbed on the outside of the cell membrane and simulated potassium-channel proteins via bulk of carboxyl for K+permeating.The secondary structure and conformation of aromatic amino acids in the tertiary structure of Cyt c remained unchanged after the interaction with CNPs.Cyt c was found to be absorbed on the surfaces of CNPs in a non-linear manner and only bound Cyt c can be reduction from oxidation state to reduction state.The results of cyclic voltammetry showed that the CNPs had little effect on the redox potential of Cyt c.The CNPs were proved to be biocompatible with BY-2 cells in the cell viability assay.There were no obvious differences in cellular ATP and the mitochondrial membrane potential(MMP)after exposure to CNPs only when the CNPs concentration was below 31.25 μg/mL.The levels of reactive oxygen species(ROS)increased at high concentration of CNPs(≥31.25 μg/mL)in BY-2 cells.Production of H2O2 and MDA and antioxidase activities were reduced with increasing concentration of CNPs in BY-2 cells.Evaluation on cytotoxicity showed that the entry of CNPs did not cause serious oxidative stress effect on the defense system of BY-2 cells.In addition,there was negative correlation between K+accumulation and H2O2 production,as well as CAT activity.However,there was no correlation between K+accumulation and MDA production,as well as antioxidase activities(SOD,POD,and APX).Analysis of metabolites content revealed that there were significant differences between the low(control,0,6.25 and 31.25 μg/mL)and high concentration CNPs groups(62.50 and 125.00 μg/mL).These results provide new insights into the relationship between the promotion of potassium uptake and the underlying toxicity induced by CNPs in BY-2 cells.