The Role And Mechanism Of NLRP3 Inflammasome In Toxic Heavy Metal-induced Inflammatory Response

BackgroundNLRP3 inflammasome is a cytosolic protein complexe that is formed to mediate host immune responses to microbial infection and cellular damage.With a various pathogens,particulate matters and injuries stimulation,NLRP3 inflammasome triggers proteolytic cleavage of dormant procaspase-1 into active caspase-1,and converts the cytokine precursors pro-IL-1? and pro-IL-18 into mature and biologically active IL-1? and IL-18,which contribute to the inflammatory response.Excessive inflammatory response leads to immune dysfunction,cell death,tissue injury,and associated with the pathogenesis of various inflammatory diseases.Nickel oxide nanoparticles(NiONPs),a representative type of metal oxide nanoparticle,are manufactured for various applications,such as catalysts,ceramics,storage batteries,fuel cells,sensors and paint formulations.The increasing production and application of Ni ONPs has led to an increase in the environmental burden and a serious hazard to human health.Cadmium(Cd)is a widespread and extremely toxic industrial and environmental pollutant that endangers human health.The pollution and hazard of Cd has been a hot issue in the toxicology and environmental medicine.Aberrant inflammatory response play a crucial role in toxic heavy metal-induced celluar and tissue damage.Therefore,this study was conducted to investigate the role and mechanism of NLRP3 inflammasome activation in Ni ONP induced pulmonary inflammation,and to test the hypothesis that melatonin could confer hepatoprotection via inhibiting TXNIP-mediated NLRP3 inflammasome activation.Methods(1)The dult male Sprague Dawley rats were administrated with 3.3 mg/kg Ni ONP by intratracheal instillation.Total cell numbers,neutrophil numbers,total protein concentrations,LDH and ALP in bronchoalveolar lavage fluid(BALF)were determined at 3 days,7 days,and 28 days postexposure.Hematoxylin and eosin staining was applied to examine pathological changes of lung tissue.NLRP3 inflammasome activation was detected by western blot after Ni ONP exposure.(2)The cellular uptake and cytotoxicity of NiONP were performed by Newport Green staining,cell viability and LDH release aasays in RAW264.7 and BEAS-2B cells.Subcellular localization of uptaked Ni ONP was observed by TEM.The effects of Ni ONP on lysosome function were examined.The role of phagolysosome acidification and cathepsin B in NiONP-induced NLRP3 inflammasome activation and cellular damage were investigated using vacuolar proton-ATPases inhibitor(BafilomycinA1)and cathepsin B inhibitor(CA-074Me),respectively.(3)The cell viability and LDH release aasays were applied to evaluate the cytotoxicity of Cd on primary cultured hepatocyte.Parallel,the cell viability,LDH release,ROS production and inflammatory cell death were conducted to evaluate the potential protective effects of melatonin against Cd-induced hepatic injury and inflammation.The effects of melatonin pretreatment on Cd-induced TXNIP expression and NLRP3 inflammasome activation were detected by western blot.The role of TXNIP-NLRP3 inflammasome pathway in the protective effects of melatonin against Cd-induced hepatotoxicity was investigated by si RNA against TXNIP.Results(1)Intratracheal instillation of NiONP resulted in sustained pulmonary inflammation and injury.Ni ONP exposure increased ALP activity,LDH levels and total protein concentrations in BALF,and induced inflammatory cell infiltration,alveolar proteinosis and cytokine secretion in lung tissue.NiONP activated NLRP3 inflammasome in rat lungs.(2)After uptake by RAW264.7 and BEAS-2B cells,Ni ONP inhibited cell viability and increased LDH release in a dose-and time-dependent manner.Ni ONP induced cytokines secretion and NLRP3 inflammasome activation.Knockdown of NLRP3 or inhibition of caspase-1 abolished Ni ONP-induced NLRP3 inflammasome activation and IL-1? secretion.Disruption of actin-mediated phagocytosis completely suppressed the NiONP-induced cytotoxicity,NLRP3 inflammasome activation and IL-1? production.Uptaked NiONP were translocated to lysosome and decreased lysosomal protease activity,increased lysosomal p H level,lysosomal membrane permeabilization and cathepsin B release.Inhibition of phagolysosome acidification and cathepsin B release reduced NiONP-induced NLRP3 inflammasome activation and IL-1? secretion.(3)Cd exposure induced a time-dependent hepatotoxicity.Melatonin pretreatment significantly alleviated Cd-induced cytotoxicity by suppressing ROS production,decreasing TXNIP expression and inhibiting NLRP3 inflammasome activation.Knockdown of TXNIP abolishes melatonin-inhibited NLRP3 inflammasome activation in Cd-treated primary hepatocytes.ConclusionNiONP induced persistent pulmonary inflammation and injury via NLRP3 inflammasome activation.Ni ONP exposure induced phagolysosome acidification and lysosome membrane permeabilization,which then results in cathepsin B release.The elevated cytoplasmic cathepsin B cause NLRP3 inflammasome activation,leading to inflammation and tissue injury.Furthermore,our results suggested that melatonin confers protection against Cd-induced liver inflammation and hepatocyte death via inhibition of the TXNIP-NLRP3 inflammasome pathway.This study indicates that melatonin may be a promising pharmacological candidate for antagonizing toxic heavy metal-induced inflammatory response.