Effects And Mechanism Study Of Exogenous Pb Exposure On Autophagy-lysosomal Pathway In PC12 Cells

Heavy metal lead(Pb)contamination in food affects the quality and safety of food.Lead has neurotoxicity and damages the central nervous system,but its toxic mechanism is not fully understood.Autophagy-lysosomal pathway is an important catabolic lysosomal degradation process essential for homeostasis and cell survival.Dysfunctional autophagy-lysosomal pathway has been associated with a wide range of human diseases.At present,there is no systematic study on the effect of Pb exposure on autophagy-lysosomal pathway.Objective: 1.To investigate the effect of Pb exposure on autophagy-lysosomal pathway and analyse the the characteristics of this effect.2.To explore the mechanism of Pb exposure on autophagy-lysosomal pathway through the entire process of autophagy.Methods: 1.We used PC12 cells as the neurocellar model.The MTT reduction assay was used to investigate the toxic effects of Pb exposure on cell viability of PC12 cells.2.Western Blot was applied to assay the protein level of autophagy-related protein LC3 in Pb-exposed PC12 cells.3.The number of autophagosomes in Pb-treated PC12 cells was assayed by Western Blot and fluorescence microscopy analysis of GFP-LC3.4.The fluency of autophagic flux was examined by the protein level of SQSTM1/p62 in Pb-exposed PC12 cells.5.The effect of Pb exposure on the formation of autophagosomes was evidenced by treated PC12 cells with 3-MA(autophagy inhibitor)and sh RNA against ATG5 in the presence or absedce of lead and the the protein level of Beclin1.6.The effect of Pb exposure on the autophagosomal-lysosomal fusion was assayed by the colocalization coefficient of LAMP1 and GFP-LC3.The relative m RNA level of Stx17,Vamp8,SNAP-29 was assayed by fluorescence quantitative PCR.7.The protein level of LAMP1(a lysosomal marker)was assayed by Western Blot.Lyso Tracker Red staining and Lyso Sensor Green DND-189 staining were used to detect the cytosolic lysosomes and lysosomal p H.Results: 1.The cell viability of PC12 cells was significantly reduced by Pb exposure in a dose-dependent manner.2.The protein level of LC3-II was increased by Pb exposure and this change suggested the accumulation of autophagosomes in PC12 cells.3.The autophagosome accumulation was caused by the inhibition of autophagic flux and the blockade of autophagy due to Pb exposure was time-dependent and reversible.4.Pbdid not affect the activation of autophagy and the formation of autophagosomes.5.Pb inhibited the fusion between autophagosomes and lysosomes in PC12 cells and the impaired autophagosomal-lysosomal fusion was not related with the expression of SNARE complex.6.Pb limited the number or size of lysosomes and also alkalinized lysosomal p H which impaired lysosomal function.Conclusion: These data suggest that Pb impaired lysosomal density and function which caused the blockade of autophagosome-lysosome fusion in PC12 cells,resulting the inhibition of autophagic flux.The dysfunctional autophagy may contribute to Pb-induced neurotoxicity in PC12 cells.