Research On The Role Of Mitophagy In Silver Nanoparticle-induced Hepatocytotoxicity

BackgroundSilver nanoparticles(AgNPs)are one of the most commonly used nanomaterials and are increasingly being incorporated into consumer products and medicines.These nanoparticles are potentially harmful to human health and have aroused widespread concern.The liver is one of the organs most sensitive to both acute and chronic AgNP exposure.AgNPs can damage liver tissue,induce cell dysfunction,and lead to liver dysfunction.Therefore,it is important to study the toxic effects of AgNPs on the liver.Mitochondria are sensitive targets for NP-induced cytotoxicity.Mitochondrial dysfunction plays an important role in cell fate.The regulation of the hepatotoxicity of AgNPs has become a key mechanism in safety studies regarding AgNPs.Studies have found that AgNPs can damage mitochondrial structure and function and induce an oxidative stress response.Mitochondrial dynamics and mitophagy,as important mechanisms for maintaining cellular function may also be involved in the toxic effects induced by AgNPs.However,there are limited studies on the role and molecular mechanism of the regulatory relationships among mitochondrial dynamics,oxidative stress and mitophagy in hepatotoxicity induced by AgNPs.Therefore,an in-depth mechanism study and target regulated factors exploring among these bioprocesses are importance to clarify the interactions between AgNPs and liver cells.ObjectivesThe purpose of this study was to systematically reveal role and molecular mechanism of mitophagy in the AgNP-induced hepatotoxicity;the potential relationship between AgNP-induced mitochondrial dynamics imbalance/oxidative stress interaction,mitophagy and mitochondria-dependent apoptosis were evaluated carefully.In addition,the role of silver ion(Ag⁺)in the AgNP-induced hepatotoxicity were explored.Contents and methods1.Silver nanoparticles and characterization.Transmission electron microscopy(TEM),flow cytometry,UV-Vis's spectrophotometry,and a Malvin particle size analyzer were used to observe polyvinylpyrrolidone(PVP)-coated AgNP particle morphology.2.Hepatotoxic effects of AgNPs.ICR mice were injected with different concentrations of AgNPs.The effects of AgNPs on body weight,liver function,histopathology,and inflammatory factors were evaluated.Analysis of silver content in liver tissues by ICP-MS.CCK-8 method was employed for cytotoxic effects of different doses of AgNPs were studied in HepG2 cells.The uptake of AgNPs in HepG2 cells was detected by flow cytometry and ICP-MS.In vivo and in vitro experiments were performed to assess indicators of mitochondrial damage(including changes in mitochondrial membrane potential,morphology,structure,and reactive oxygen species(ROS)production).Western blotting was used to detect the effects of AgNP exposure on mitochondria-dependent apoptosis signaling pathways.3.To investigate the role of dynamin-related protein 1(DRP1)-mediated mitochondrial fission in the hepatotoxic effects induced by AgNPs.Immunofluorescence and western blotting were used to detect the effects of AgNP exposure on mitochondria fission.To clarify the role of mitochondrial division in mitochondrial dysfunction and apoptosis induced by AgNPs,the effects of DRP1 deficiency on AgNP-mediated mitochondrial damage,oxidative stress,and mitochondria-dependent apoptosis were assessed in HepG2 cells.4.To investigate the role of oxidative stress in the hepatotoxicity induced by AgNPs.To elucidate the role of oxidative stress in mitochondrial dysfunction,mitochondrial fission and apoptosis induced by AgNPs in hepatocytes,the antioxidant N-acetylcysteine(NAC)was used to evaluate the role of oxidative stress in mitochondrial dysfunction,DRP1-dependent mitochondrial fission,and mitochondria-dependent apoptosis.5.To investigate the role of PTEN-induced kinase 1(PINK1)/Parkin-regulated mitochondrial autophagy activation in the cytotoxic effect of HepG2 induced by AgNPs.In vitro and in vivo experiments,firstly,the role of AgNPs in mitophagy and autophagy activation of hepatocytes was explored by immunofluorescence and western blotting.Secondly,DRP1 siRNA,or mitochondrial division inhibitor 1(Mdivi-1),and NAC were used to determine the relationship between DRP1-mediated mitochondrial fission,oxidative stress,and mitophagy/autophagy.Finally,the effects of PINK1 deficiency on mitochondrial dysfunction,mitochondrial fission,oxidative stress,and mitochondria-dependent apoptosis in the hepatotoxicity after AgNP exposure were assessed.Results1.TEM images indicated that PVP-coated AgNPs were well dispersed and spherical,with an average diameter of 23.44±4.92 nm.The ultraviolet absorption spectrum showed an absorption peak for AgNPs at 400-420 nm.The results of Malvin particle size analyzer showed that PVP-coated AgNPs had certain agglomeration in different media.2.12 and 120 mg/kg AgNPs were deposited in the liver tissues of ICR mice,causing liver dysfunction,histopathological changes,increased release of pro-inflammatory factors and hepatic dysfunction.However,exposure to 5 mg/kg AgNO₃,equivalent to the silver content of12 mg/kg AgNPs,did not induce significant toxic effects on mouse hepatocytes.In vitro studies have also confirmed that HepG2 cells have the ability to take up AgNPs.AgNP(20-160?g/mL)-induced decreased MMP,ROS overproduction,and increased apoptosis in a dose-dependent manner in HepG2 cells.However,exposure to 1.2?g/mL AgNO₃,which was equivalent to 160?g/mL AgNPs in silver content,did not induce significant toxic effects on HepG2 cells.3.Exposure to AgNPs promotes mitochondrial division in hepatocytes.In vivo,12 and 120mg/kg AgNPs exposure in a dose-dependent manner induced mitochondrial structural changes and induced mitochondrial dynamics imbalance,mainly due to the increased expression of key protein p-DRP1 of mitochondrial fission in mouse hepatocytes.In vitro,the mitochondrial structure and morphology of HepG2 cells were induced after exposure of 160?g/mL AgNPs for 48 h.After exposure to 20-160?g/mL AgNPs for 48 h,HepG2 cells induced an imbalance of mitochondrial dynamics in a dose-dependent manner,mainly due to the increased expression of p-DRP1,a key protein for mitochondrial fission.Inhibition of DRP1-dependent mitochondrial fission can reduce mitochondrial damage,oxidative imbalance and mitochondria-dependent apoptosis induced by AgNPs in HepG2 cells.In vitro and in vivo AgNO₃ exposure did not significantly affect the mitochondrial dynamic balance of hepatocytes.4.Exposure to AgNPs induces oxidative stress in hepatocytes.In vivo,after exposure to 12and 120 mg/kg AgNPs,the oxidative stress related protein heat shock protein 70(Hsp70)and heat shock proteins 70(Hsp70)and heme oxygenase 1(HO-1)was induced in a dose-dependent manner in mouse hepatocytes.In vitro,after exposure to 20-160?g/mL AgNPs for 48 h,ROS production and the expression levels of Hsp70 and HO-1 in HepG2 cells were increased in a dose-dependent manner.In addition,NAC verified that inhibition of oxidative imbalance can also alleviate mitochondrial fission and mitochondria-dependent apoptosis induced by AgNPs.5.Exposure to AgNPs induces mitophagy in hepatocytes.In vivo,12 and 120 mg/kg AgNPs exposure in a dose-dependent manner induced increased expression levels of mitophagy-related proteins PINK1 and Parkin in mouse hepatocytes.In addition,after AgNPs exposure,autophagosomes accumulated,and the expression levels of autophagy marker protein microtubule-associated protein 1 light chain 3B(LC3B)and autophagy substrate p62 were increased.In vitro,the protein expression levels of PINK1,Parkin,LC3B and p62 in HepG2cells were increased in a dose-dependent manner after exposure to 20-160?g/mL AgNPs for48 h.After 160?g/mL AgNPs exposure for 48 h,there was also obvious accumulation of autophagosomes in HepG2 cells.In vivo and in vitro,AgNO₃ exposure did not activate mitophagy or autophagy in hepatocyte.DRP1 inhibition and the use of the antioxidant NAC confirmed that DRP1-dependent mitochondria fission/oxidative stress is a component of the upstream mechanism that activate mitophagy.By inhibiting PINK1 expression,we verified that PINK1/Parkin-mediated mitophagy reduced mitochondrial fragmentation,MMP collapse,ROS overproduction,oxidative/reductive imbalance,and elevated mitochondria-dependent apoptosis in HepG2 cells treated with AgNPs.ConclusionIn the present study,we confirmed that AgNPs particles,rather than Ag⁺,induce hepatocytotoxicity effects in vivo and in vitro,and verified that AgNP-induced mitochondrial dysfunction and mitochondrial-dependent apoptosis.The crosstalk between DRP1-dependent mitochondrial fission and oxidative stress also activates mitochondrial-dependent apoptosis,mitophagy and autophagy.Importantly,mitophagy plays a protective role in AgNP-induced hepatotoxicity by inhibiting mitochondrial fission,oxidative stress,and mitochondria-dependent apoptosis.These results provide basic laboratory data for comprehensive evaluation of hepatotoxicity induced by AgNPs,and also provide sensitive targets for reducing hepatotoxicity of AgNPs.