Mechanism Of Benzo(a)pyrene Induced Lipid Accumulation By Autophagy Inhibition And Screening Of Nutritional Intervention Agents

Disorders of lipid metabolism will lead to the occurrence of various system diseases,such as hyperlipidemia,diabetes,atherosclerosis,etc.Benzo(a)pyrene(Bap)is a food processing contaminant.Its sources are: industrial discharge affects the soil and contaminates food indirectly;second,food is inappropriately fried,smoked,cooked,grilled,etc.Benzo(a)pyrene has lipotoxicity,teratogenicity,immunotoxicity and genotoxicity.According to reports,long-term exposure of benzo(a)pyrene can induce abnormal accumulation of cellular lipids,which is extremely harmful to the human system.Autophagy is a regulatory mechanism in lysosomes of eukaryotic cells.It maintains the homeostasis of the cell environment mainly by lysosomes degrading damaged organelles,abnormally folded proteins,and cell debris.When it is interfered by the outside world,autophagy will happen to realize the self-repair of the body.The study found that benzo(a)pyrene can induce autophagic damage to cells,but the understanding of autophagy signal transduction and its effect on cell survival is not yet comprehensive,and the mechanism of autophagy needs further study.In recent years,a large number of experiments have proved that abnormal lipid accumulation is closely related to autophagy dysfunction.Therefore,the main purpose of this study is to explore the effect of benzopyrene on lipid accumulation and autophagy disorder in RAW264.7 cells,and to elaborate on the specific mechanism and screening of nutritional intervention agents.The main contents and results of this paper are as follows:(1)Study on benzo(a)pyrene Induced Lipid Accumulation in RAW264.7 Cells.The non-toxic dose of benzopyrene screened by thiazole blue(MTT)experiment was0-40 ?M.Benzo(a)pyrene could induce lipid accumulation by Oil Red O staining and total cholesterol(TC)and triglyceride(TG)content detection.Benzopyrene-induced lipid accumulation was related to the inhibition of autophagy by adding the autophagy activator Rapamycin(Rapa)and the inhibitor chloroquine(CQ).(2)Benzo(a)pyrene inhibits autophagy of RAW264.7 cells and mechanism.Detection of LC3 protein expression determined that benzo(a)pyrene significantly affected autophagy at a concentration of 40 ?M at 6 h by Western Blot method.Observation through transmission electron microscopy showed that 40 ?M benzo(a)pyrene significantly accumulated autophagosomes.It was determined that benzo(a)pyrene inhibited autophagy and blocked the flow of autophagy by adding3-methyladenine(3-MA),chloroquine(CQ)and m RFP-GFP-LC3 adenovirus transfection.Through immunofluorescence staining,LAMP-2 protein expression and lysosomal p H detection,it was determined that benzo(a)pyrene promoted the fusion of autophagosomes and lysosomes,resulting in an increase in lysosomal p H and a decrease in degradation ability.(3)Improvement effect of nutritional intervention agents on benzo(a)pyreneinduced lipid accumulation.The non-toxic doses of Salidroside,Allicin,and Alliin were selected by MTT experiment as 0-100 ?M,0-50 ?M and 0-1600 ?M.Through oil red O staining,TC,TG content detection,it was determined that the three intervention agents at 50 ?M can significantly reduce intracellular lipid droplets,TC and TG content,and play a role in lipid-lowering.Through the detection of lysosomal p H and c AMP activity,it was determined that the three nutritional intervention agents can effectively improve the lysosomal damage and increase the activity of c AMP.In summary,this study observed that benzo(a)pyrene can induce lipid accumulation in RAW264.7 cells.It was found that benzo(a)pyrene can inhibits cell autophagy and blocks the flow of autophagy,revealing that alkalization of the lysosomal environment is the reason for the blocking of autophagy flow.It was determined that nutritional intervention agents such as salidroside,allicin,and alliin can improve the benzo(a)pyrene-induced lipid accumulation,and provide experimental evidence and data support for the exposure hazard of benzo(a)pyrene and the establishment of anew control plan.It has Important theoretical and practical significance.