Effects Of Inhibiting RROS/NLRP3/Caspase-1 Mediated Pyroptosis On Cadmium-induced Apoptosis Of Duck Renal Tubular Epithelial Cell

Cadmium(Cd),a heavy metal pollutant,enters the body and is widely distributed in various organs and tissues,mainly in the kidneys and liver.In severe cases,it can cause liver and kidney damage,pyroptosis,and cancer.The purpose of this study was to investigate the effect of Cd on pyroptosis of duck renal tubular epithelial cell and its relationship with apoptosis.The culture model of primary renal tubular epithelial cell of ducklings was established by enzyme digestion in 10-15-day-old ducklings.The cell were cultured with3Cd SO₄·8H₂O(0,2.5,5.0,or 10.0?mol/L Cd),N-acetyl-L-cysteine(NAC)(100?mol/L),Z-YVAD-FMK(10.0?mol/L)and the combination of Cd and NAC or Z-YVAD-FMK for12 h and then cytotoxicity was assessed.After 12 h,biochemical analysis,morphological analysis,determination of reactive oxygen species(ROS)level,changes in mitochondrial membrane potential(MMP),apoptosis rate,m RNA expression of pyrolysis-related genes and apoptosis-related genes,and Caspase-1 p20,ASC,NLRP3,GSDMD,p53,Bax,Bcl-2,cleaved-Caspase-9,Caspase-3,cleaved-Caspase-3 protein expression.The results are as follows:1.Compared with the control group,the content of cadmium in the cell and supernatant of each Cd treatment group increased significantly in a concentration-dependent manner(P<0.001),indicating that the accumulation of intracellular Cd increased with the increase of Cd concentration.2.Compared with the control group,the Cd group can significantly increase the release and relative conductivity of lactate dehydrogenase(LDH),nitric oxide(NO),IL-18,IL-1?,and intracellular reactive oxygen species(ROS)levels.Cd can up-regulate NLRP3,Caspase-1,ASC,NEK7,IL-1?,IL-18 m RNA levels and Caspase-1 p20,NLRP3,GSDMD,ASC protein levels in a dose-dependent manner,especially 10?mol/L Cd is the most obvious(P<0.001).3.Compared with Cd group,the biochemical indexes(release of IL-18,IL-1?,LDH and NO),relative conductivity,intracellular ROS level,m RNA level of pyroptosis-related genes(NLRP3,Caspase-1,ASC,NEK7,IL-1?,IL-18)and protein level(Caspase-1 p20,NLRP3,GSDMD,ASC)in NAC+Cd group decreased significantly(P<0.05,P<0.01 or P<0.001),indicating that NAC can reduce the pyroptosis of duck renal tubular epithelial cell induced by Cd.4.Compared with the control group,the YVAD treatment group significantly inhibited the expression of Caspase-1 m RNA and protein,while the expression of NLRP3did not change significantly.Compared with the 10?mol/L Cd group,the YVAD+Cd group can significantly reduce the expression of Caspase-1 and NLRP3 genes and proteins,and reduce the content of LDH,NO,IL-1?and IL-18 in the supernatant,and Conductivity level.It shows that YVAD has a significant inhibitory effect on Caspase-1.5.Compared with Cd group,YVAD+Cd group increased MMP(P<0.001),decreased apoptosis rate and cell injury,significantly increased Bcl-2 m RNA level(P<0.001),and decreased the expression of p53 Bax,Bak-1,Caspase-9,Caspase-3,Cyt C m RNA(P<0.001 or P<0.001)and p53 Bax,Bak-1,cleaved-Caspase-9,Caspase-3.Cleaved-Caspase-3 protein levels(P<0.05,0.01 or P<0.001).Taken together,Cd exposure could induce duck renal tubular epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway,and inhibiting pyroptosis of Caspase-1dependence might weaken Cd-induced apoptosis.