The Effect Of Cadmium On Autophagy And Lysosomal Function In Mouse Liver Cells And The Protective Effect Of Puerarin

The heavy metal cadmium(Cd)is a highly ubiquitous environmental pollutant that can be transported into organisms by breathing and eating the contaminated substance.The accumulation of Cd can cause acute and chronic damage in human and animals.Notably,liver is one of the momentous target organs that damaged by Cd.Previous studies have shown that Cd exposure could induce autophagy,which is a protective mechanism against Cd-induced cellular injury in hepatocyte.Puerarin(Pue),a flavonoid glycoside purified from the root of the plant Puerara lobate,has multiple biological activities,such as scavenge oxygen free radicals,antioxidant,anti-cancer,regulate blood sugar,enhance immunity,exc.Researches have shown that Pue could induce autophagy and protect cellular injury induced by Cd,but the concrete mechanism remains unclear.In order to study the effect of Cd and Pue on autophagy and lysosome function,and the effect of autophagy on Cd-induced hepatic cell injury,the AML 12 cells were treated with Cd and Pue.Then the effect and the potential mechanisms of the process above were studied by using cellular molecular biology techniques.1.The protective effect of puerarin on Cd-induced cell injury in AML12 cellsIn order to study the protective effect of Pue on Cd-induced cell injury,AML12 cells were treated with Cd alone or in combination with Pue.Then the cell indexes were monitored by RTCA,the cell morphology and nuclear morphology were viewed under inverted fluorescent contrast phase microscope,the ultrastructure of nucleus and mitochondria were viewed under transmission electron microscopy.The results showed that the rise velocity of cell index were inhibited,and the nuclear morphology as well as the cell morphology were changed after Cd exposure.Moreover,the ultrastructure of nucleus and mitochondria were damaged due to Cd exposure.In addition,Pue play a protective role on Cd-induced cell injury in MALI 2 cells.2.The effect of Cd on autophagy in AML 12 cells and the protective effect of puerarinIn order to study the effect of autophagy on Cd-induced cell injury and the protective effect of Pue,AML 12 cells were treated with 5μM Cd alone or in combination with 200 μM Pue for 12 h.After treatment,transmission electron microscope,MDC fluorescence staining and immunofluorescence were used to study the level of autophagy in AML12 cells.Besides,AML12 cells were treated with varying concentrations of Cd(0,2.5,5,10,20μM)for 12 h or 5μM Cd for varying time durations(0,3,6,12,24 h),then examined the autophagy related proteins levels by western blotting.Moreover,AML12 cells were treated with 5 μM Cd alone or in combination with 50 nM Baf or 200 μM Pue for 12 h separately,then examined LC3 and P62 protein expression levels by western blotting to monitor the autophagic flux.Furthermore,AML12 cells were treated with or without 50 nM RAPA or 50 nM Baf before treated with 5μM Cd,then monitor the cell index by RTCA.The results showed that the autophagy level increased after Cd exposure and reached the highest level(P<0.01)as cells were treated with 5μM Cd for 12 h.Cd exposure blocked the autophagic flux,which aggravated the cell injury in AML12 cells.Pue induced autophagy and promoted the autophagic flux in AML12 cells,which would be conducive to mitigating the Cd-induced cell injury.3.The effect of Cd on lysosomal function in AML12 cells and the protective effect of puerarinIn order to study the possible mechanism of the Cd-induced autophagy blockage and the protective effect of Pue in MALI 2 cells,cells were treated with varying concentrations of Cd(0,2,5,5,10,20μM)for 12 h or 5μM Cd for varying time durations(0,3,6,12,24 h),and cells were treated with 5μM Cd alone or in combination with 200μM Pue for 12 h.After treatment,the pH of lysosome was examined by LTR fluorescence staining,the levels of lysosome-associated proteins were examined by western blotting,and the degradation function of lysosome were reflected by monitoring the degradation of DQ-BSA.In addition,cells were treated with 200μM Pue,50 nM Baf,or 50 nM TG before treated with 5μM Cd,then observed the co-localization of LC3 with LAMP2 in AML12 cells.The results showed that Cd triggered lysosomal activation demonstrated by increased LTR staining,increased the expression of CTSB and LAMP2.What’ more,the DQ-BSA-Green staining ware increased,but,the co-localization of LC3 with LAMP2 were decreased dramatically after Cd exposure.Besides,the expression of Rab7 ware decreased,moreover,showed a significant dose-effect and time-effect correlation.Interestingly,Pue further enhanced the lysosomal activation,and recovered the expression of Rab7.Conclusion:Cd induced cell injury and promoted autophagy in AML12 cells.Moreover,Cd blocked the autophagic flux,which aggravated the cell injury in AML 12 cells.Cd triggered lysosomal activation and promoted the lysosomal function in protein degradation,but inhibited the fusion of autophagosomes and lysosomes.In addition,Cd decreased the expression of Rab7 that a key protein for the fusion of autophagosome and lysosome.Furthermore,Pue decreased Cd-induced cell injury related to it induced autophagy in AML12 cells.And Pue recovered the fusion of autophagosomes and lysosomes as well as the expression of Rab7,which remitted the autophagy blockade induced by Cd in AML12 cells.