Effects Of Inhibiting Pyroptosis On Apoptosis Of Duck Renal Tubular Epithelial Cell Induced By Excess Molybdenum

Molybdenum（Mo）is one of the essential trace elements in organisms,which involves in many physiological and biochemical activities.However,excessive Mo can induce Mo poisoning in the body.The kidney is an important place for body metabolism and a target organ for Mo toxicity.Pyroptosis and apoptosis are two types of programmed cell death.To investigate the impacts of excess Mo on pyroptosis and the relationship between pyroptosis and apoptosis in duck renal tubular epithelial cells.The cells were treated with[（NH₄）₆Mo₇O₂₄·4H₂O]（0,480,720 and 960μmol/L Mo）,N-acetyl-L-cysteine（NAC）（100μM）,Z-YVAD-fluoromethylketone（YVAD）（10.0μM）and the joint of Mo and NAC or YVAD for 12 h.The concentration of Mo in cells and cell supernatant were detected inductively coupled plasma mass spectrometry;The levels of pyroptosis,apoptosis-related genes and proteins were detected by real-time quantitative PCR and western blot;The microplate reader was used to detect the release of IL-1βand IL-18 nitrogen monoxide（NO）,lactate dehydrogenase（LDH）in cell supernatant;The relative conductivity of the supernatant was detected by conductivity meter;The changes in mitochondrial membrane potential（MMP）and levels of intracellular ROS were measured with fluorescence microscope;The apoptosis rate was detected by fluorescence microscope.The results are as follows:1.Excessive Mo notably upregulated Caspase-1,NLRP3,ASC,NEK7,GSDMA,GSDME,IL-1β,IL-18 mRNA levels and Caspase-1 p20,NLRP3,ASC,GSDMD protein levels and the changes of 720μM Mo group were the most obvious.Simultaneously,excess Mo also markedly increased LDH,NO,IL-18,IL-1βrelease,relative conductivity,cell average area and intracellular ROS level.2.Correlation analysis showed that ROS levels was strongly positive correlated with pyroptotic genes mRNA levels.Besides,compared with the 720μmol/L Mo group,the co-treatment of NAC and 720μmol/L Mo could significantly attenuate Mo-induced the changes of above these indicators.3.Compared with the control group,the levels of Caspase-1 mRNA and protein were markedly reduced in the YVAD group,and NLRP3 had no significant different.Compared with the 720μmol/L Mo group,the co-treatment of YVAD and 720μmol/L Mo could significantly reduce the expression of Caspase-1 and NLRP3 genes and proteins.YVAD also could markedly improve Mo-induced the changes of LDH,NO,IL-1β,IL-18 release,relative conductivity,cell average area,and ROS level.4.Compared with the 720μmol/L Mo group,YVAD and 720μmol/L Mo co-treatment could dramatically decrease the cell apoptosis rate and increase the MMP.5.Compared with the 720μmol/L Mo group,YVAD and 720μmol/L Mo co-treatment could significantly decrease Caspase-3,Bak-1,Bax,cytochrome C（Cyt C）mRNA and cleaved-Caspase-3 protein expression levels,decrease the levels of Bcl-2mRNA as well as the ratio of Bcl-2/Bax.Conclusion:Excessive Mo exposure can induce pyroptosis by the ROS/NLRP3/Caspase-1 pathway in duck renal tubular epithelial cells,and restraining pyroptosis of Caspase-1 dependence might weaken excess Mo-induced apoptosis.The study provides theoretical basis for excess Mo exposure nephrotoxic research on waterfowl and the interplay between pyroptosis and apoptosis highlights a new sight into the mechanism of Mo-induced nephrotoxicity.