Carbon Black Nanoparticles And Lead Ions Co-induce NR8383 Alveolar Macrophage Apoptosis And Potential Mechanisms

Background:Airborne particulate matter（PM）is the main component of air pollution affecting human health,and its toxic effects have been intensively studies.Among PM,ultrafine particles,nanoparticles,have a relatively specific larger surface area and can absorb large amounts of toxic pollutants.Such nanoparticles are easily enter and accumulate into the lungs,and can cause airway epithelial and other types of cell injury and damage.However,how nanoparticles absorbed with toxic pollutants alter their physical characteristics and whether nanoparticles absorbed with toxic pollutants can modulate their toxicity against alveolar macrophages have not been clarified.This study aimed to explore the physicochemical characteristics and cytotoxicity of Carbon black nanoparticles absorbed with Pb²⁺against alveolar macrophages and its potential mechanism in vitro.CBNPs are an excellent model for studying airborne particulate toxicity.CBNPs in the air are mainly derived from soot produced by incomplete combustion of the combustion source.Due to its small particle size,it can penetrate deeply into the alveoli and directly affect lung function.Lead(Pb²⁺)is a typical air pollutant derived from human activities,such as fuel combustion,industrial production and waste incineration.Both of them can enter the lung through the respiratory tract and can accumulate in the lungs,promoting the development of respiratory diseases.Alveolar macrophages are located in the alveolar space and tiny airways.They are crucial for cleaning toxic pollutants and defensing invaded micro-pathogens.During the pathogenic process of pulmonary diseases,macrophage damage is one of the important mechanisms.In this study,we employed CBNPs and Pb²⁺in a cellular model of rat alveolar macrophages（NR8383）to explore the cytotoxicity of CBNPs and Pb²⁺and its potential mechanisms in vitro.Objective:This study aimed at generating CBNPs-Pb²⁺nanoparticles and understanding their physiochemical characteristics;investigating the cytotoxicity of CBNPs and Pb²⁺against NR8383 cells,and exploring whether CBNPs and Pb²⁺-caused oxidative stress to modulate autophagic flow,leading to apoptosis of NR8383 cells in vitro.Methods:1.Particle characterization:CBNPs and CBNPs-Pb²⁺were characterized by DLS,TEM,SEM and multi-point BET analysis.ICP-OES was used to quantify the adsorption of Pb²⁺by CBNPs and to assess the stability of the infected particles.2.Cytotoxicity studies:Rat alveolar macrophages（NR8383）were treated with different concentrations of CBNPs and/or Pb²⁺for 24 h,and thier cell viability was analyzed.The cell apoptosis was determined by flow cytometry after Annexin V-FITC/PI staining,fluorescent microscopy using DAPI staining,and western blot.The levels of reactive oxygen species（ROS）production and mitochondrial membrane potential in individual groups of cells were analyzed by flow cytometry after DCFDA and JC-1 treatment,respectively.3.Mechanistic studies:The levels of autophagic influx was examined by transmission electron microscopy（TEM）,mRFP-GFP-LC3 adenovirus-based confocal fluorescent microscopy and Western blot following treatment with autophagy inducer of rapamycin or the late stage of autophagy inhibitor of chloroquine（CQ）.Results:1.CBNPs-Pb²⁺nanoparticles were successfully established.CBNPs effectively and stably adsorbed Pb²⁺,and CBNPs-Pb²⁺nanoparticles displayed an altered physicochemical properties of Pb²⁺.The dynamic light scattering indicated that CBNPs-Pb²⁺nanoparticles reduced the average particle size and dispersed for a period of time after sonication.TEM revealed that CBNPs-Pb²⁺nanoparticles were more effectively phagocytosed by alveolar macrophages cells.2.CBNPs-Pb²⁺significantly reduced the viability of NR8383 cells but increased the frequency of apoptotic NR8383 cells.3.CBNPs-Pb²⁺significantly increased ROS levels and decreased mitochondrial membrane potential in NR8383 cells,leading to cell apoptosis;4.CBNPs-Pb²⁺significantly enhanced Caspase 3 and Caspase 9 activation,and increased Bax expression,but decreased anti-apoptotic Bcl-2 expression,leading to an decreased in ratio of Bcl-2/Bax in NR8383 cells.5.CBNPs-Pb²⁺inhibited the autophagic influx,resulting in an accumulation of autophagosomes in cells,but reduction in autophagolysosomes and lysosomal dysfunction in NR8383 cells.6.Treatment with CQ,an autophagolysosomal inhibitor,enhanced the effect of CBNPs-Pb²⁺on autophagic influx and cell apoptosis,while treatment with rapamycin,an inhibitor of mTOR and inducer of autophagy,mitigated the effect of CBNPs-Pb²⁺on autophagic influx and cell apoptosis in NR8383 cells.Conclusion:Our data indicated that CBNPs-Pb²⁺had cytotoxicity against NR8383 cells by triggering their apoptosis.Our study found that CBNPs could adsorb Pb²⁺uniformly and stably,and the physicochemical properties of the adsorbed nanoparticles changed.The cytotoxic effects of CBNPs-Pb²⁺were from that 1)CBNPs-Pb²⁺induced oxidative stress and damaged the mitochondrial membrane and function;2)inhibited autophagic influx and decreased lysosomal function.Our findings may provide new insights into understand the pathogenesis of toxic pollutant nanoparticle-induced pulmonary diseases and aid in design of new strategies for prevention and intervention of these diseases.