Study On The Cell Death Mediated By Carbon Nanoparticles In Roasted Chicken Breast Retained In Lysosome

Carbon dots are carbon nanoparticles with fluorescent properties.Carbon nanotubes,carbon fibers,graphite rods,carbon ash,and activated carbon can be used to synthesize carbon quantum dots by means of arc discharge,laser ablation,electrochemical synthesis,and heat treatment.Thermo process is one of the important ways to prepare carbon quantum dots.Thermo process is also the most commonly method in food processing.It can produce carbon dots after the food being baked by heat treatment.The higher the baking temperature of the food,the smaller diameter and the greater the toxicity of carbon dots on cells.The chicken breast was used as the research object.After high-temperature roasting,we extracted and purified carbon dots to study the mechanism of carbon dots-induced cell death.This study will provide a theoretical guidance for the roasting and processing of chicken meat and a reference for the research on the biological effects of endogenous carbon nanoparticles in other thermally processed foods.It can prevent the harm of food-borne carbon nanoparticles to human health and provide the foundation for improving food safety.The main research results of the full text are as follows:1.Extraction and identification of carbon spotsIn this paper,the endogenous carbon nanoparticles produced by roasting chicken breasts at high temperature were extracted and purified.The transmission electron microscopy data showed that and the carbon dot has a nearly spherical structure and the particle size is about 6 nm.X-ray diffractometer(XRD)data showed that the carbon dots were in an amorphous state.Fourier infrared spectroscopy(FTIR)results showed that the surface groups of carbon dots were methyl,methylene and aromatic hydrocarbon.X-ray photoelectron spectroscopy(XPS)data showed that the elements of carbon dots were C,N,and O.The high-resolution C_1S spectrum results showed that the chemical bonds on the surface of the carbon dots were C=O,C-O/C-N,and O-C=C.The N_1S high-resolution spectrum results showed that the presence of pyrrole N and pyridine N in the carbon dots.2.The study on the mechanism of carbon dot-induced cell deathThe carbon dots were treated with NRK cells for 12 h.The bright field and transmission electron microscopy results showed that the carbon dots internalized in the lysosomes of the cells.We found that carbon dots can induce apoptosis in NRK by the method of flow cytometry.The Lamp-1-cherry NRK cells were treated with carbon dots for 12 h and the lysosomal morphology was captured by SP8 Leica confocal microscopy at a Z-axis interval of 0.1?M.Thirty-layer images were scanned,and then reconstructed in three dimensions by image processing software Hygens 19.04.Our results showed that carbon dots can increase the size of lysosomes.The results of DQ-red BSA confirmed that the intensity of red fluorescence was reduced in carbon dot treated group.The change of lysosomal p H in cells was tested after carbon dot treatment by the method of flow cytometry with lysosensor DND189,a lysosomal p H detection probe.The results showed that the carbon dots increased the lysosomal p H.Necroptosis is one of the most common death pathways of cell death.It is regulated by kinases receptor interaction protein 1(RIPK1),receptor interaction protein 3(RIPK3)and mixed lineage kinase domain-like protein(MLKL).Our results showed that the key regulatory gene RIPK1 and RIPK3 can co-localize with lysosomes.Moreover,the protein levels of RIPK1 and RIPK3 in the carbon dot treatment group were higher than those in the control group.Our results confirmed that the carbon dots in roasted chicken breasts damaged the lysosomal membrane,changed the permeability of the lysosomal membrane,increased the p H of the lysosome and induced lysosomal-dependent cell death.Carbon dots caused the increase of permeability of lysosomal membranes and aggregation of RIPK1 and RIPK3proteins in lysosome,induced lysosome-dependent cell death and amplified the death signal via necroptosis.