The Effects Of Autophagy And Pyroptosi In The Process Of Liver Injury Induced By Benzo(?) Pyrene

Liver is the biggest parenchymatous organ in people's enterocoelia,and liver injury is the primary reason of hepatic disease.The liver injury which caused by the harmful ingredients in food(such as benzo(?)pyrene)can not be ignored,and he research for hazardous substance in food can provide scientific basis for preventing liver disease.As a common polycyclic aromatic hydrocarbon(PAHs)compounds which is produced in the processing of oil and fats products,benzo(?)pyrene exists widely in our daily lipid foods.Benzo(?)pyrene is not only a type of strong cancerogen,but also has fortissimo immunotoxicity and reproduction toxicity.It can cause body injury and damage the human health through the way of induced cell DNA damage and mitochondrial dysfunction,oxidative stress.Cell autophagy is the basic stress response of cells that ensure their survival under harsh conditions.It mainly through the degradation of their own abnormalities or excessive organelles and proteins to achieve their own metabolism:and regeneration balance.Cell autophagy imbalance will induce cell death.The pyroptosi is a cell death that relies on caspase-1 activation,which in turn is associated with the release of a large number of pro inflammatory cytokines.However,the mechanism of liver damage caused by benzo(?)pyrene is not clear.The effects of benzo(?)pyrene on the cell viability,autophagy,and pyroptosi of HL-7702 cells were explored in this thesis at the cellular level using benzo(?)pyrene as research object and select HL-7702 hepatic cell as model.The relationship between cell autophagy and pyroptosi induced by benzo(?)pyrene in human HL-7702 cells also was investigated through adding the inhibitor of autophagy and pyroptosis.The main findings are as follows:(1)The effects of benzo(?)pyrene on the viability of HL-7702 hepatic cell were researched through those methods of MTT test,staining test,flow cytometry analysis,and fluorescence probe detection of DCFH-DA.The result indicated that the proliferation of HL-7702 hepatic cell was inhibited significantly by benzopyrene in a time and concentration-dependent manner.The cell viability was significantly decreased to 71%and 75%after 50 pM benzo(?)pyrene treatment on HL-7702 hepatic cell for 24 h and 48 h respectively.It was obviously to discern that the cell mortality rate was increases with the concentration of benzo(?)pyrene gradually increases through the trypan blue staining test after benzo(a)pyrene treatment on HL-7702.The experimental result of FCM analysis displayed that the cell cycle of HL-7702 hepatic cell was blocked in S period by benzo(?)pyrene,the amount of later period apoptotic cell and the reactive oxygen in cell increased significantly.Therefore the benzo(?)pyrene could inhibited the proliferation of HL-7702 hepatic cell through the way of blocking cell cycle,inducing apoptosis and increasing reactive oxygen.(2)The effects of benzo(?)pyrene on the autophagy damage of HL-7702 hepatic cell were studied by fluorescence staining and transmission electron microscopy.The results of AO and MDC fluorescence staining test showed that the fluorescence spots intensity of AO and MDC in HL-7702 hepatic cell treated by benzo(?)pyrene enhanced with the concentration increase of benzo(?)pyrened gradually.The results of TEM also showed that the amount of autophagic vacuoles in HL-7702 hepatic cell increased gradually when the treatment concentration of benzo(?)pyrene increased,and the phenomenon of the autophagic vacuole phagocytosis the Golgi organelle was observed when the concentration of benzo(?)pyrene was increased to 50 ?M.At the same time,the expression quantity of Beclin-1 and LC-3 in HL-7702 hepatic cell increased significantly after the cell treatmented with benzo(?)pyrene and the expression quantity showed a dose-dependent manner.So the experimental result demonstrated that HL-7702 hepatic cell was induced by benzo(?)pyrene could generated autophagy damage and the degree of autophagy had a positive correlation with the concentration of beno(?)pyrene.(3)The effects of benzo(?)pyrene on the pyroptosis of HL-7702 hepatic cell were studied by flow cytometry.The relative conductivity,NO release quantity and LDH release rate of HL-7702 hepatic cell were significantly increased after treatment with benzo(?)pyrene.At the same time,the expression quantities of Caspase-1?COX-2,IL-1?,IL-18 were improved significantly in HL-7702 hepatic cell after benzo(?)pyrene treatment,and the expression quantities showed a dose-dependent manner.This research proved that the pyroptosis could be induced by benzo(?)pyrene in HL-7702 liver cells,and the degree ofpyroptosis was positively correlated with the concentration of benzo(?)pyrere.(4)The relationship between cell autophagy and pyroptosis induced by benzo(?)pyrene in HL-7702 hepatic cell was further investigated by Western-blot and other methods.Adding autophagy inhibitor of 3-Methyladenine,the relative electrolytic leakage,the relative conductivity,NO release quantity and LDH release rate of HL-7702 hepatic cell were significantly decreased compared with those cells that only treatment with benzo(?)pyrene,and the expression quantity of Caspase-1,COX-2,IL-1?,IL-18 also decreased significantly.Adding pyroptosis inhibitor of Ac-YVAD-CMK,the results of AO dye experiment showed that the fluorescence intensity and the expression quantity of Beclin-1,LC3 in HL-7702 hepatic cell decreased significantly compared with those cells that only treatment with benzo(a)pyrene.The results of the above experiments indicated that that the cell pyroptosis induced by benzo(a)pyrene was inhibited when the autophagy was inhibited in HL-7702 hepatic cell induced by benzo(?)pyrene.Meanwhile,the cell autophagy induced by benzo(a)pyrene was inhibited when the pyroptosis induced by benzo(a)pyreneis in HL-7702 hepatic cell also was inhibited.In a word,cell pyroptosis and autophagy was induced simultaneously by benzo(?)pyrene in HL-7702 hepatic cell,and the two actions promoted each other.The effects of benzo(?)pyrene on cell viability,autophagy and pyroptosis in HL-7702 hepatic cell were studied in this research,and the relationship between cell autophagy and pyroptosis was deeply explored.All the results will provide a scientific basis for the revealing of the the toxicity mechanism of benzo(?)pyrene,and the establishment of efficient precautionary measures.