The Underlying Mechanisms Of Atmospheric PM_2.5 Disturbing The Autophagy In Endothelial Cells And Protective Effects Of Allicin

Objective:1)In order to provide the theoretical basis for the scientifically assess the potential risks of atmospheric PM_2.5 exposure on human cardiovascular system,the underlying molecular mechanisms of atmospheric PM_2.5 disturbing human umbilical vein endothelial cells?HUVECs?autophagy were studied.2)The protective effects and possible molecular mechanisms of allicin on HUVECs due to atmospheric PM_2.5 exposure were explored to provide new ideas and strategies for improving the cardiovascular health of residents affected by atmospheric PM_2.5 exposure.Methods:?1?In the present study,the transmission electron microscope?TEM?was employed to observe the distribution of PM_2.5 in endothelial cells and the formation of autophagosomes in the HUVECs treated with 1,5,25?g/mL concentrations of PM_2.5 for 24 h.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?.The proteins expression of LC3 A,LC3 B,Beclin 1,p62,ATG3,ATG7,ATG5,ATG12,STX17,CTSB,CTSD,LAMP1,LAMP2 were determined with western blot.AKT,ERK1/2 and TFEB signaling pathways were selected to explore the potential molecular mechanisms.?2?The cytotoxicity were determined with the levels of released lactate dehydrogenase?LDH?from HUVECs,induced by PM_2.5 and three concentrations?5,10,20?g/mL?of allicin.Cell morphology and apoptotic bodies were observed with Hoechst 33258 staining,and the flow cytometry was employed to determinate the apoptosis of HUVECs exposed to the PM_2.5 and allicin.The TEM was employed to observe autophagosomes of HUVECs.The levels of LC3 and p62 puncta were detected by IF.The ELISA was employed to investigate the levels of inflammatory cytokines?IL-1?,IL-4,IL-6,IL-8,TNF-??,chemokines?MCP-1?and adhesion molecules?VCAM-1,sICAM-1?.The proteins expression and activation of TF,PAI-1,Bcl-2,Bax,caspase-3,caspase-9,PARP,p62,Beclin 1,LC3 II/I,Nrf2,HO-1,keap 1,NF-?B and MAPK signaling pathway?ERK1/2,p38,JNK1,JNK2?were detected by western blot.Results:?1?The results showed that the entrapment of PM_2.5 into HUVECs,it was mainly located in the small capsule with membrane wrapped in the cytoplasm,indicating that PM_2.5 mainly entered the cell through cellular swallowing and then produced toxic effects on the cell.The number of autophagosomes and LC3 puncta,and the levels of proteins?LC3 II/I,Beclin 1,ATG3,ATG7,ATG5,ATG12?all were increased in HUVECs exposed to PM_2.5,indicating that PM_2.5 increased the number of autophagic vesicles in HUVECs.In addition,the increasing levels of p62puncta and protein in the exposed groups indicated that the contents of the autophagic vesicle?'cargo'?were not effectively degraded.The number of lysosomes,and the levels of LAMP1 and lysosomal marker protease CTSB and CTSD all increased in the exposed group,as well as the increasing phosphorylation of upstream regulators ERK1/2 and TFEB,indicating enhanced lysosomal activity.Moreover,the levels of STX17and LAMP2 proteins of HUVECs decreased with the elevating levels of PM_2.5 exposure,showing autophagosome and lysosome fusion was blocked.Meanwhile,phosphorylation of AKT,an upstream regulator of autophagy,increased in the exposed group,which inhibited the autophagy process by activating the mTORC1 protein complex.In summary,these results suggested that PM_2.5 exposure inhibited the formation of autophagic vesicles and their fusion with lysosomes by activating AKT,thus blocking the normal autophagic flux of HUVECs.?2?In the allicin and PM_2.5 exposed group,allicin could significantly reduce the activation of LDH and the apoptotic rate of HUVECs,compared with the PM_2.5 group.Moreover,the increasing ratio of Bcl-2/Bax,and reducing activation of the downstream proteins caspase-3,caspase-9 and PARP,were also observed with allicin administration in the western blot,indicating that allicin could protect of PM_2.5-induced damage of HUVECs in a certain extent and reduced apoptosis.At the same time,the expression of TF and PAI-1 were reduced,the decreasing proinflammatory factor?IL-1?,IL-6,IL-8,TNF-?,MCP-1,VCAM-1,sICAM-1?,and decreasing the levels of phosphorylation of NF-?B,while the increasing anti-inflammatory factor of IL-4,indicating that allicin inhibited the cellular inflammatory response induced by PM_2.5 through inhibiting the activation of NF-?B.In addition,the number of autophagosomes,LC3 and p62 puncta,and the levels of proteins?p62,Beclin 1,LC3 II/I?were all decreased of HUVECs,which treated with allicin and PM_2.5,compared with the PM_2.5 group,indicating that allicin could restore the interference of PM_2.5 on autophagy of endothelial cells.On the contrary,compared with the PM_2.5 single exposure group,increasing the levels of phosphorylation of Nrf2,up-regulated of HO-1,down-regulated of keap 1 of HUVECs treated with allicin and PM_2.5,indicating that allicin could improve PM_2.5 induced endothelial cells oxidative damage.After being exposed to both allicin and PM_2.5,enhancing activation the intracellular MAPK signaling pathway,including the levels of phosphorylation of ERK1/2,p38,and JNK,compared with the PM_2.5 single exposure group.To sum up,allicin increased the phosphorylation of Nrf2 and inhibited the activity of NF-?B through activating MAPK,and participated in the regulation of endothelial cell apoptosis,inflammation,autophagy and oxidative stress by PM_2.5,thereby improving dysfunction of endothelial cells.Conclusions:?1?PM_2.5 decreased the expression of STX17 and LAMP2 by activating AKT,inhibited the fusion of autophagosome and lysosome,and blocked the normal autophagic flux of HUVECs.?2?Allicin up-regulated Nrf2 phosphorylation,inhibited NF-?B activity,regulated intracellular apoptosis,inflammation,autophagy and oxidative stress by activating MAPK signal pathway,so as to improve endothelial cell dysfunction induced by PM_2.5 to some extent.