Protective Effect Of Trehalose On Cadmium-induced Cellular Damage In Rat Renal Tubular Epithelial Cells

Cadmium(Cd)is a widespread environmental contaminant in our country.It accumulates in humans and animals via food chain through the water,plants,animals and other ways.It exerts toxic effects on multiple organs in mammals,nearly 50% of Cd accumulated in the renal proximal tubules.Trehalose(Tre)is a newly discovered autophagy enhancer and its mechanism of action in antagonizing heavy metal Cd has not been elucidated.Therefore,Tre act as a protective agent explored the protective effect of Cd-induced death in rat renal tubular epithelial cells(rPT cell)from three perspectives: apoptosis,autophagy,and oxidative stress in this study.Firstly,primary cultured cells were incubated with Cd and Tre.It was found that 5 mM Tre was chosen to evaluate its protective effect on Cd-induced cytotoxicity through CCK-8assay and morphological changes.Next,we detect apoptotic rate and cell morphological changes by flow cytometry and laser confocal microscopy.In addition,we detect the expression of cleaved-PARP,cleaved-caspase 9 and cleaved-caspase 3 proteins by western blotting.We found that Tre could down-regulate apoptotic rate and level of apoptotic key protein.These data reveal that Tre could inhibit Cd-induced apoptosis activation and cell death.Second,we investigated effect of Tre on Cd-induced autophagy inhibition.It was found that Tre could significantly alleviate Cd-induced inhibition of autophagic flux and reduce the accumulation of autophagosomes via analyzing the changes in the expression of LC3 and SQSTM1/p62 protein and combining with the results of RFP-GFP-LC3 and GFP-LC3 transfection.Next,we found that Tre can alleviate the Cd-induced autophagosome-lysosomal fusion blocked through detecting the autophagosome-lysosome fusion(LAMP-1 and LC3co-localization)by laser confocal microscopy.In addition,the lysosomal acidity and lysosomal degradability were measured.The results showed that Tre could restore Cd-induced lysosomal function damaged,thus restoring the ability of lysosome.Finally,we explored the effect of Tre on Cd-induced oxidative stress.The results showed that Tre can alleviate Cd-induced oxidative stress in rPT cells by detecting level of reactive oxygen species(ROS)and malonaldehyde(MDA).We further investigated whether Tre isrelated to the alleviation of Cd-induced oxidative damage,because the Nrf2-Keap-1 pathway is an important oxidative stress defense mechanism in cells.It was found that Cd could activate Nrf2 into the nucleus and enhance the level of the downstream Nrf2 related antioxidant proteins through detecting the anti-oxidation proteins in the Nrf2-Keap-1 related pathway,such as oxidoreductase 1(NQ-O1),heme oxygenase-1(HO-1),glutathione synthesis-related proteins(GCLC,GCLM,and GR)and related detoxification enzymes(SOD,CAT and GPx).However,Tre could inhibit Nrf2 activation and down-regulated the level of relevant antioxidant proteins.These data iluustrrated that Tre could alleviate Cd-induced oxidative damage and inhibit Nrf2-Keap-1 pathway activation.In summary,Tre is a novel autophagy enhancer that plays an important role in the protection of Cd-induced cell death.First,Tre could alleviate Cd-induced apoptosis through inhibiting mitochondrial apoptosis pathway.Second,Tre reduces autophagic flux,promotes autophagosome-lysosomal fusion and relieves lysosomal damage,thus alleviating Cd-induced autophagy inhibition.In addition,Tre as an antioxidant can alleviate Cd-induced oxidative stress,mainly by inhibiting Cd-induced Nrf2-Keap1 signaling pathway.In short,we provide a theoretical basis for Tre to reduce Cd-induced cell death via three aspects of apoptosis,autophagy and oxidative stress in this study.