Study On The Damage Effect Of CML On Human Brain Vascular Endothelial Cells And Its Mechanism Based On 3D Cell Model

BackgroundsMaillard reaction can easily produce the potential hazards of glycosylation(AGEs)in food processing.Carboxymethyl lysine(CML)is an AGE marker with clear structure and is widely found in food.Therefore,humans are at risk of exposure to high concentration of foodborne CML.CML studies on physical hazards are still at the stage of accumulation of raw data,and no conclusion has been reached yet.Furthermore,the mechanism of toxic effects of food-derived CML in vivo has not been analyzed at the molecular level.Human body tissue is three-dimensional(three-dimensional,3D)structure,but the researchers often rely on the two-dimensional(two-dimensional,2D)culture in vitro and animal models in vivo for human tissue structure,function and pathological study.Using normal 2D cell culture to study the development mechanism of tumor development is very different from the real environment,such as cell and matrix interaction,cell differentiation and so on.Animal models cannot reappear human characteristics well,such as human tumor growth and metastasis,drug therapy,immune response,etc.Therefore,this study intends to use human brain vascular endothelial cells(HBVEC)to construct a 3D microvascular network model in vitro to study the effect and mechanism of CML on human brain tissue.MethodsThe HBVEC cells were co-cultured with the hydrogel and perfused into a microfluidic chip to construct a 3D microvascular network model.The formation process of the microvascular network was photographed using a laser confocal microscope.Verify the fluidity and aggregation of the 3D cell model.The HBVEC cells were divided into five groups and cell culture medium containing final concentrations of 0.05 mg/mL,0.1 mg/mL,0.2 mg/mL,and 0.5 mg/mL of CML was added for the exposure.24 hours after exposure,CCK-8 kits was used to determine the effect of CML on the viability of HBVEC cells;GSH-Px and ROS kits were used to determine the oxidative damage of HBVEC cells by CML.The expression of IL-6,TNF-?,VCAM,MCP-1,RAGE,AP-1,NF-?? and P38 mRNA was measured by real-time fluorescence quantitative PCR.Results1.Compared with 2D cell model,3D cell model can more effectively simulate the intercellular microenvironment in vivo.This model has good reproducibility and stability when evaluating the cytotoxic effect of CML on HBVEC.2.CML has certain toxic effects on HBVEC cells and causes oxidative damage to cells.Cell activity decreased as the concentration of CML increased,and the final viability decreased by 14%.ROS increased with the increase of CML concentration and increased by 71% to the greatest degree.CML also has an effect on GSH-Px activity.3.CML may cause oxidative damage to the cerebral blood vessels,thereby breaking the blood-brain barrier into the brain.Fluorescent quantitative detection of the expression of related genes revealed abnormalities such as upregulation of P38 gene,indicating that CML can promote the expression of P38 and other inflammatory cytokines.ConclusionThe use of human brain vascular endothelial cells to construct a 3D microvascular network model can well mimic the migration and transport of CML in human tissues.CML can induce inflammation by inducing oxidative stress and immune response to human brain vascular endothelial cells,and has obvious toxic effects on cells.