Protective Effect Of Puerarin On Cadmium-induced Lysosomal Dysfunction In Rat Proximal Tubular Cells

Cadmium(Cd)is a toxic heavy metal that is widely exist in the environment.It's widely used in industrial production.Cd is a highly accumulated poison,which has a long biological half-life and serious injury on body.It can produce a strong toxic effect on the organs.The kidney is the main organ of the accumulation of Cd,especially the renal cortex.It's already proved that puerarin(PU)has a protective effect on lead-induced rat proximal tubular cells damage,but the protective effect of PU on Cd-induced rat proximal tubular cells was not reported.Therefore,PU was used as an antagonist of Cd in this experiment.The protective effects of PU were discussed from three aspects: autophagy,lysosomal and oxidative stress.An in vitro model of primary rat proximal tubular cell(rPT)was established in this study.Cells were treated with different concentrations of PU for 12 h during the period of Cd exposure.Firstly,the cell viability was detected by CCK-8 and the morphology changes of cells were observed by phase contrast microscopy.It was found that the best protective concentration of PU is 100 ?M.Secondly,investigated whether PU can restore the inhibition of autophagy that induced by Cd.The changes of the expression level of autophagy marker proteins LC3-? and p62 were detected by immunoblotting technique.In combination with the results of transient transfection of RFP-GFP-LC3 and GFP-LC3 plasmid,showed that PU treatment can attenuated Cd-induced the fusion block of autophagosome and lysosome and the accumulation of autophagosome.The results demonstrated that PU can alleviate the autophagy inhibition in Cd-induced rPT cells.Thirdly,in view of the key role of lysosomes in autophagy,the effect of PU on on lysosomal function was discussed.The internal environment of lysosomes was detected by Lyso-Tracker-Red and AO staining respectively.The results showed that PU significantly attenuated Cd-induced lysosomal pH alkalization.The DQ-BSA fluorescent staining and the protein expression level of CTSD revealed that PU can significantly alleviate Cd-induced lysosome degradation declining.In combination with the results of Lysoo-Tracker-Red and AO staining,the results of colocalization of LAMP-1and CTSD indicated that PU effectively prevented the lysosomal membrane permeabilization(LMP)induced by Cd.These results showed that the lysosomal dysfunction induced by Cd can be restored by PU.Fourthly,the effect of PU on oxidative stress on Cd-induced rPT cells was discussed.The levels of ROS and MDA and the changes of DHE fluorescence intensity in the intracellular were detected by flow cytometry and chemical colorimetric method and laser confocal microscopy technique,respectively.The results showed that PU treatment can significantly alleviate oxidation stress caused by Cd.To further confirm the protective effect of PU on Cd-induced rPT cells,the nuclear translocation of Nrf2 and the expression levels of Keap1 and Nrf2-Keap1 pathway downstream signal molecules proteins were detected.It was found that PU significantly inhibited the activation of the Nrf2 nuclear translocation and the activation of Nrf2-Keap1 pathway induced by Cd.At the same time,PU treatment significantly reduced the expression levels of antioxidant enzymes and GSH-synthesis-related proteins in Cd-induced rPT cells.These results further confirmed that PU significantly alleviated the oxidative stress in Cd-induced rPT cells.In summary,PU can alleviate autophagy inhibition,restore lysosomal dysfunction and attenuate the activation of Nrf2-Keap1 pathway in Cd-induced rPT cells.The antioxidant properties of PU plays an important role in Cd-induced autophagy inhibition and lysosomal functional damage.This study provides a basis for the further development of PU as a novel therapeutic strategies for Cd-induced nephrotoxicity.