| Cadmium（Cd）is a kind of toxic heavy metal which exists widely in the ecological environment.It causes multi-organ damage to the body.Kidney is one of the target organs of Cd toxicity,and renal tubule is the target site of Cd-induced kidney injury.Previous studies found that 2.5 μM Cd induced oxidative stress in rat renal tubular epithelial cells and led to apoptosis.It has been reported that 10 μM Cd exposure activated NLRP3 inflammasome,increased the m RNA expression of pro-inflammatory cytokines,causing inflammatory response,but the specific molecular mechanism remains unclear.Necroptosis is a new type of cell death mediated by RIPK1-RIPK3-MLKL.Similar to traditional necrosis,necroptosis can cause rupture the plasma membrane,release DAMPs,and thus contributing to inflammatory response.MLKL is a key executive protein of necroptosis,which mediates membrane rupture through localization to cell membrane or organelle membrane.Whether it is involved in Cd-induced intracellular inflammation remains unclear.Therefore,in this study,the role of necroptosis in Cd-induced inflammatory response in NRK-52 E cells was investigated in vitro,and the specific mechanism of MLKL as necrotic executive protein to induce inflammatory response was revealed.Rat renal tubular epithelial cells（NRK-52 E cells）were treated with Cd,and a series of biological indicators were detected by related reagents.CCK-8 and LDH release assay showed that NRK-52 E cells treated with 10 μM Cd for 24 h significantly reduced cell survival rate and increased LDH release.Flow cytometry with Annexin-V-FITC/PI double staining was performed and necrotic cells were significantly increased in 10 μM and 20 μM Cd treatment groups.Ultrastructural changes of the cells were observed under transmission electron microscopy.It was found that the cells treated with 10 μM Cd were swollen and deformed with membrane rupture,nucleolysis,and mitochondrial crest blurred or even disappeared,showing typical characteristics of necrosis.Meanwhile,the expression levels of RIPK3 and MLKL increased in a dose-dependent manner.After silencing MLKL gene,cell death rate was significantly alleviated,further confirming that NRK-52 E cell death induced by 10 μM Cd was necroptosis.The detection results of inflammation related indicators（NLRP3 inflammasome,HMGB1,pro-inflammatory cytokines TNF-α,IL-1β,IL-2,IL-6 and IL-18 m RNA levels）showed that,Cd exposure activated NLRP3 inflammasome and HMGB1,and then promoted the transcription and maturation of inflammatory cytokines,which induced intracellular inflammatory response.Since damaged mitochondria can produce excessive ROS to aggravate intracellular inflammation,and transmission electron microscopy results also showed that mitochondria were damaged in the Cd treatment group,it is necessary to further explore the effects of mitochondrial damage on intracellular inflammation caused by Cd.The expression levels of mitochondrial fission proteins（Drp1 and Fis1）were up-regulated and the expression levels of fusion proteins（OPA1 and Mfn1）were down-regulated by 10 μM Cd exposure,and the morphology of mitochondria changed into scattered and abscissive point structures.In addition,mitochondrial membrane potential and ATP decreased significantly,and the intracellular ROS level increased significantly.Mdivi-1 pretreatment inhibited the changes of Cd-induced mitochondrial biological indicators and ROS accumulation,suggesting that the imbalance of mitochondrial dynamic homeostasis contributed to Cd-induced mitochondrial dysfunction and ROS production.However,NAC pretreatment significantly alleviated Cdinduced intracellular inflammatory response via removing excessive ROS.In view of the special role of MLKL as the executive protein of necroptosis,the mitochondrial localization of MLKL was investigated.Firstly,this study found that Cd induced MLKL translocation to mitochondria and interaction with Drp1,disrupting mitochondrial function,while gene silencing MLKL significantly reduced the co-localization between MLKL and mitochondria,alleviating the imbalance of mitochondrial dynamic homeostasis and mitochondrial dysfunction in NRK-52 E cells.In addition,MLKL knockdown inhibited Cd-induced m PTP opening,mitochondrial calcium loss and reduction of GSH and NADPH content in antioxidant system,and thus preventing ROS accumulation in NRK-52 E cells.These results suggested that Cd-induced co-localization of MLKL with mitochondria plays an important role in ROS accumulation,which mediated inflammatory response in NRK-52 E cells.In conclusion,Cd induces RIPK3-MLKL dependent necroptosis in NRK-52 E cells,and activated MLKL plays a role in the imbalance of mitochondrial dynamic homeostasis via targeting mitochondria,and a large number of ROS produced by damaged mitochondria induce intracellular inflammatory response.Meanwhile,Cd-induced the mitochondrial localization of MLKL disrupted mitochondrial function thruogh interaction with Drp1,causing mitochondrial calcium loss,which is unable to provide power for the NADP+/NADPH antioxidant enzyme system,and leads to the decrease of GSH content and ROS accumulation in NRK-52 E cells,ultimately resulting in the inflammatory response.Therefore,this study elucidates the mechanism of necroptosis in Cd-induced inflammatory response in NRK-52 E cells,suggesting that MLKL may serve as a new therapeutic target for antagonizing Cd-induced renal tubular epithelial cell injury. |
