The Mechanism Of Toxicity Research Of The Silver Nanoparticles (Ag-NPs) On The Rat Glioma Cells Line RG2

ObjectiveGlioma is the most common primary tumor of the central nervous system.It is a neuroepithelial tumor with high invasiveness,poor prognosis and high postoperative recurrence rate.At present,there is no effective treatment for this disease.The malignant growth of glioma is based on the enhancement of proliferation ability and the damage of cell differentiation and apoptosis.The apoptosis inhibitory factor bcl-2 and the apoptosis-activating factor caspase-3 play a key role here.As a new antitumor drug,Ag-NPs has stable physical and chemical properties,obvious penetration and retention effects of tumor tissue,strong surface modification and loading properties,and good biocompatibility.However,whether Ag-NPs has cytotoxic effect on the rat glioma cells line RG2 or not,and the mechanism of Ag-NPs's cytotoxicity to RG2 cells is not clear.Therefore,a model of cytotoxicity of Ag-NPs to RG2 cells in vitro was established to Preliminary study the mechanism of cytotoxicity of Ag-NPs on glioma.MethodIn this experiment,the study of RG2 cells was carried out in vitro.RG2 cells were inoculated into 96 well and 6 well culture plates,and cytotoxic killing model of tumor cells was established by adding different concentration gradient Ag-NPs?0,0.0125,0.025,0.05,0.1,0.2 mg/mL?into the culture plate.Ag-NPs was characterized by UV-Vis spectrophotometer and TEM,and its morphology,size and dispersion were investigated.The effect of Ag-NPs on the proliferation of RG2 cells was detected by CCK-8 assay.The effects of Ag-NPs on the size,morphology and organelle of RG2cells were observed by optical microscope and TEM,and the location of Ag-NPs in RG2 cells was determined by TEM.The effect of Ag-NPs on intracellular ROS level and cell cycle was detected by immunoflow cytometry.The effect of Ag-NPs on apoptosis of RG2 cells was determined by Annexin V-FITC double staining.Western blot technique was used to detect the effect of Ag-NPs on the expression of caspase-3and bcl-2 in RG2 cells.ResultThe experimental results showed that the peak of Ag-NPs absorption spectrum was409.5 nm,spherical,uniform in size,and the average particle size was 20.06±2.57 nm,which could be evenly dispersed in the medium.Ag-NPs can enter RG2 cells by endocytosis and is located in lysosomes and vesicles.AgNPs can promote RG2 cells to produce cell membrane rupture,mitochondria vacuolization,chromatin nuclear membrane edge polymerization,apoptotic bodies and autophagic corpuscles.Ag-NPs can significantly inhibit the proliferative activity of RG2 cells,CC₅₀=0.575mg/mL.The cytotoxicity was concentration-dependent,and the degree of inhibition was positively correlated with the concentration of AgNPs.Ag-NPs could significantly increase the level of ROS in RG2 cells and promote oxidative stress in a concentration-dependent manner.Ag-NPs can induce RG2 cells to block in S phase of cell cycle,resulting in DNA synthesis obstacle.Ag-NPs could induce apoptosis and necrosis of RG2 cells,up-regulate the expression of Caspase-3,and inhibit the expression of bcl-2 protein.ConclusionAg-NPs has obvious toxic effects on RG2 cells.The mechanisms include inhibition of cell proliferation,enhancement of oxidative stress response in cells,destruction of cell membrane and enhancement of membrane permeability,damage of DNA and arrest of cell cycle,and apoptosis and necrosis of RG2 cells were induced.