Dynamin-related Protein 1(Drp1)-RB Axis Regulates Mitochondria-dependent Necroptosis Of Hepatocytes Induced By Cadmium

Necroptosis,one of regulated cell death(RCD),involves the activation and initiation of receptor interacting protein kinase(RIPK)1 and RIPK3,and recruitment the mixed lineage kinase domain-like(MLKL)protein forms a necrosome complex that causes damage of the membranes of cell and organelles.Necroptosis plays an important role in pathological changes such as inflammatory,infectious,and degenerative diseases as well as acute and chronic liver injury.Cadmium(Cd)and its compounds(Cd2+)are a common heavy metal contaminant,mainly from industrial production,such as mining,alloy manufacturing,electroplating,and rechargeable batteries.The pathways that enter the body include Cd-contaminated water,air,and food.In addition,smoking is another major source of Cd exposure to population.Accumulation of Cd in the body can cause damage to many tissues and organs such as the liver and kidney.The previous study found that the endoplasmic reticulum-autophagy pathway mediates toxicity and damage of Cd.Another study indicates that mitochondria are important targeted organelle for Cd damage,and their components,structure,and function are controlled by mitochondrial quality control(MQC)system.Dynamin-related protein 1(Drp1)triggers mitochondrial fission-mediated mitochondrial homeostasis,which is an important part of MQC.Retinoblastoma(RB)protein can be directly targeted to mitochondria and is not dependent on transcriptional regulation to control MQC.MQC monitors mitochondrial integrity at the molecular,organelle,and cellular levels and regulates necroptosis mediating many body damages caused by environmental exposure,including Cd.Therefore,it is of great significance to investigate the mechanisms and interventions of Cd in the regulation of hepatic toxicity and injury via MQC-necroptosis through relevant protein interactions.Objectives:This study aims to investigate the hepatotoxicity and molecular mechanisms of Cd-induced necroptosis,and to explore the regulatory mechanisms of MQC disorder,up-regulation and mitochondrial translocation of RB protein-mediated necroptosis,in order to provide intervention targets and strategies for mitochondria-associated hepatotoxicity induced by xenobiotics.Methods:(1)In vivo evaluation of liver injury induced by exposure to cadmium chloride(CdCl2):adult male ICR mice were randomly divided into two groups including control(Ctrl,normal saline)and CdCl2 treatment(1 mg/kg·bw).Continuous intraperitoneal injections were conducted for one week and the body weight changes were recorded daily.Twenty-four hours after the end of the exposure,mice were sacrificed for liver tissues.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissues.Western blot(WB)was used to detect necroptosis-related proteins(including RIPK1,RIPK3,MLKL,and p-MLKL),MQC-related proteins(including Drp1 and Mfn2,etc.)and tumor suppressor proteins(including p53 and RB,etc.).Immunohistochemistry(IHC)was used for detecting of p-MLKL,p53,RB and Drp1 tissue distribution and expression.(2)In vitro evaluation of CdC12 exposure induced necroptosis of hepatocytes:CdCl2(0-40 ?mol/L)treatment of L02 cells for 0,3,6,and 12 h was performed for concentration-and time-dependent effects analysis and selection.The mechanism study model was established after treatment with CdCl2(20 ?mol/L)for 6 h.MTT color reaction assay and flow cytometry(FCM)were used to detect cell viability.WB was used to detect the expression of necroptosis protein.The cell membrane integrity was measured by the lactate dehydrogenase(LDH)release rate.The intracellular localization of necroptosis related proteins was detected by immunofluorescence(IF)and cells ultrastructure was examined by transmission electron microscopy(TEM).(3)In vitro assay,CdCl2 exposure induces mitochondrial damage and evaluation of intervention in liver cells:detection of mitochondrial membrane potential(JC-1 probe labeling method),mitochondrial ROS(mitoSOX probe labeling method),mitochondrial calcium ion(Rhod-2AM probe labeling method)and ATP content reflect the overall level of mitochondria;intracellular distribution of MQC-related proteins was detected by isolation of the cytoplasm and nucleus or mitochondrial fractions and IF.Different intervention models were used,including the small molecule inhibitor Mdivi-1 and the drug Metformin established Drpl inhibition model,siNC and siDNMIL established Drpl knockdown model,siNC and siRBl established RB knockdown model to verify the regulatory relationship between Drpl-RB.TEM detected mitochondrial morphology.(4)Evaluation of interactions between necroptosis and MQC-related proteins:mRNA expression and correlation of related genes after xenobiotics exposed to hepatocytes using an online public gene expression database(GEO);In vitro assay,the proximity ligation assay(PLA)and co-immunoprecipitation(Co-IP)were used to evaluate the interaction of related proteins.(5)Verification of Drp1-RB complex regulates CdCl2-induced necroptosis and hepatic injury in vivo:adult male ICR mice were randomly divided into four groups including the control group(Ctrl,normal saline),CdCl2 treatment group(1 mg/kg bw),Metformin treatment group(100 mg/kg·bw)and Metformin combined with CdCl3 treatment group.CdCl2 was injected intraperitoneally and Metformin was administered by intragastric administration,continuously exposed for one week.The body weight changes were recorded every day.After the last treatment for 24 h,the mice were sacrificed to obtain liver tissue.qRT-PCR was used to detect mRNA levels of mMT1 and mMT2 in the liver;liver tissue homogenate was used to detect the activity of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)evaluating liver function;WB was used to detect the expression of necroptosis and MQC?related protein;HE staining was used to observe the damage of liver tissue;IHC was used to detect the tissue distribution and expression of RB,Drp1,and p-MLKL.Results:(1)CdCl2 induced necroptosis of hepatocytes:In vivo,compared with the control group,the protein levels of liver RIPK3,MLKL,and p-MLKL increased in CdCl2 group;the results of IHC also showed that the expression of p-MLKL was significantly increased in hepatocytes after CdCl2 exposure.In vitro experiments,the necroptosis-related protein was significantly up-regulated and mitochondrial translocation or proximity in CdCl2-treated group L02 cells.The release rate of LDH were significantly increased and ATP depletion.TEM also observed mitochondrial swelling,membrane rupture,and cell content release.(2)CdCl2-induced hepatocyte mitochondrial dysfunction and tumor suppressor protein expression in both in vitro and in vivo experiments:In vivo,p53,RB,and Drp1 were significantly up-regulated and mitochondrial translocation occurred in the CdCl2-treated group compared with the control group;IHC results indicated that the distribution of p53,RB,and Drpl was significantly increased.In vitro,the mitochondrial membrane potential(??m)decreased,mitochondrial reactive oxygen species(mitoROS)increased,mitochondrial Ca2+overload,ATP depletion,and mitochondrial hyperfractionation were observed in L02 cells treated with CdCl2.p53,RB,and Drpl were significantly upregulated and mitochondrial translocations;Drp1 knockdown or inhibition(Mdivi-1 or Metformin)reduced CdCl2-induced p53 and RB expression,blocked both mitochondrial translocations,restored mitochondrial homeostasis and function of hepatocytes.(3)Drpl-RB is involved in the formation of necroptosis complex(necrosome):GEO analysis showed exposure of human primary hepatocytes to three xenobiotics(aflatoxin B1,amiodarone,and chlorpromazine)for 14 days,the relative expression levels of RBI,DNM1L.RIPK3,and MLKL mRNA increased,and there was a positive correlation between RBI and other three genes;in vitro,the spatial location of RB and MLKL was adjacent in the CdCl2-treated group,but not p53.In vivo,the Co-IP results of liver mitochondrial fractions showed that the mitochondrial MLKL-related necrosomes contained Drp1 and RB,suggesting that Drpl-RB participates in the formation of necrosome in the CdCl2-treated group L02 cells.Drp1 and RB increased significantly in necrosome,suggesting that the interaction was enhanced.In the Drpl inhibition group and the DNM1L knockdown group or the RBI knockdown group of the L02 cells,CdCl2-induced upregulation of p-MLKL and mitochondrial translocation of MLKL were attenuated.In vivo,compared with CdCl2-treated mice,intervention of Drpl with Metformin could reduce CdCl2-induced the expression and mitochondrial translocation of Drpl and RB,up-regulation of p-MLKL,and increase of ALT and AST activities in the liver tissue of mice.Conclusions:Exposure to Cd induces activation of mitochondrial Drp1 and mitochondrial translocation of RB in liver and hepatocytes,direct interaction of RB with Drpl on mitochondria,and participates in the formation of MLKL-associated necrosome complexes,and induces hepatocyte's necroptosis.Intervention with the Drpl-RB complex inhibited Cd exposure-induced MQC disorders,hepatocyte's necroptosis,and hepatic injury.This study provides a basis for the targeted intervention of the mitochondrial Drp l-RB complex as a potential strategy to prevent and control the induction of liver toxicity and damage-related diseases by xenobiotics.