The Combinational Effect Of Polycyclic Aromatic Hydrocarbons And Lipopolysaccharide On The Autophagy Of Huvec And The Molecular Mechanism

Objective: 1)In order to provide the theoretical basis for scientifically evaluating the effect of polycyclic aromatic hydrocarbons(PAHs)on the human cardiovascular system,the impact of polycyclic aromatic hydrocarbons on autophagy of human umbilical vein endothelial cells(HUVECs)and its potential molecular mechanism.2)In order of provide the theoretical basis for scientifically evaluating the effects of environmental mixed pollutants on cardiovascular disease,the combinational effect of polycyclic aromatic hydrocarbons and lipopolysaccharides(LPS)on the autophagy and molecular mechanisms of human umbilical vein vein cellsMethods: 1)In the present study,The protein expressions of LC3 B,Beclin-1,p62,Atg5,Atg7,Atg12,STX17,CTSB,CTSD,LAMP2 in the HUVECs treated with 2.5,5,10 μmol/L concentrations of Ba P for 24 h were determined with western blot.The acidic vesicles were observed respectively by acridine orange(AO)and dansylcadaverine(MDC)staining assays.The levels of LC3 and p62 puncta were detected by immunofluorescence(IF)AKT,ERK1/2 and TFEB signaling pathways were selected to explore the potential molecular mechanisms.After treated with 2.5,5,10 μmol/L concentrations of Na P and IYP for 24 h,the expressions of autophagy-related protein were determined with western blot.The levels of LC3 and p62 puncta were detected by IF.2)The transmission electron microscope(TEM)was employed to observe the formation of autophagosomes in the HUVECs treated with 2.5μmol/L concentrations of Ba P and 5 μg/m L concentrations LPS for 18 h.The levels of LC3 and p62 puncta were detected by IF.The acidic vesicles were observed respectively by acridine orange(AO)and dansylcadaverine(MDC)staining assays.The proteins expression and activation of LC3 B,p62,Beclin-1,AKT and MAPK signaling pathway(ERK1/2,TFEB,p38,JNK1,JNK2)were detected by western blot.After treated with 2.5 μmol/L concentrations of Na P and LPS,the proteins expression of LC3 B,p62,Beclin-1 were determined by Western blot.Results: 1)After treatment with Ba P,the number of LC3 and p62 puncta,and levels of autophagy-related protein(LC3Ⅱ/Ⅰ,p62,Beclin-1,Atg5,Atg7,Atg12)all increased in exposed group.In addition,the number of lysosomes and the levels of lysosomal marker protease(CTSB,CTSD)increased.At the same time,the phosphorylation of AKT was decreased in HUVECs exposed to Ba P,while the phosphorylation of ERK1/2 and the phosphorylation of TFEB increased.The number of LC3 and p62 puncta,and the levels of autophagy-related protein(LC3Ⅱ/Ⅰ,p62,Beclin-1 all increased in Na P and IYP exposed group.2)In the Ba P and LPS exposed group,the number of autophagosomes,LC3 puncta,p62 puncta,and the levels of proteins(p62,Beclin-1,LC3B)were decreased,indicating that LPS can inhibit autophagy of HUVECs by Ba P-induced,so that the contents of autophagosomes can be degraded effectively.After being exposed to Ba P and LPS,the phosphorylation of AKT,ERK1/2,TFEB,p38,JNK1,JNK2 were decreased,compared the Ba P-exposed group.In the Na P and LPS exposed group,the levels of proteins(p62,Beclin-1,LC3B)were decreased.Conclusions: 1)PAHs induce autophagy by activating AKT/m TOR signaling pathway,while reducing the expression levels of STX17 and LAMP2,inhibiting the autophagosomes-lysosomes fusion,to inhibit the normal autophagic flux of HUVECs.2)The combinational effect of PAHs and LPS can antagonize the autophagy induced by Ba P to some extent through the MAPK signaling pathway,and restore the normal autophagy flux of endothelial cells.