The Role And Mechanism Of NLRP3 Inflammasome In J774A.1 Macrophage Inflammation Induced By Copper Oxide Nanoparticles

Copper oxide nanoparticles(CuO NPs)are one of the most widely used metal oxide nanoparticles in the 21st century,and it has been widely used in food industry and health products,agricultural production,medical treatment and other fields.CuO NPs are used as nano-fertilizer,nano-pesticides,antibacterial agent and nano-additives.However,their widely application has greatly increased the opportunities for human exposure to CuO NPs,which can enter organisms through food.Because of their small size and high surface activity,nanomaterials are easy to interact with organelles,cells,tissues and organs of organisms,and even macromolecular substances such as proteins.CuO NPs enters the human body through digestion and absorption of food through the gastrointestinal tract,which will cause functional alienation of cells and organs,thus bringing many adverse effects to the health of consumers.CuO NPs have been reported to have a strong cytotoxic and inflammatory response.Excessive inflammation can lead to chronic or systemic inflammatory disease.NLRP3 inflammasomes are molecular platforms activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as IL-1?to engage innate immune defenses.Strong associations between dysregulated inflammasome activity and human heritable and acquired inflammatory diseases highlight the importance this pathway in tailoring immune responses.NLRP3inflammasome could be activated and assembled by a variety of nanoparticles,promote the activation of caspase-1 and cleave pro-IL-1?,thereby releasing IL-1?to induce the inflammatory response.The pro-inflammatory potential of CuO NPs has been confirmed,but the specific molecular mechanism remains unclear.In this study,we aimed to investigate CuO NPs-induced NLRP3 inflammasome activation and inflammation.Part ?:J774A.1 macrophage were used as a model and exposed to different concentrations of CuO NPs(0,5,10,15,20?g/m L).Through Western-blot,RT-qPCR,ELISA,immunofluorescence and other molecular biological detection methods,the CuO NP-induced cytotoxicity and inflammatory response were explored,and the role of NLRP3 inflammasome in CuO NPs-induced cell inflammation was clarified.Firstly,CCK-8 and LDH experiments showed that CuO NPs could significantly induce cytotoxicity and damage cell membrane of J774A.1 macrophage.Furthermore,the level of IL-1?m RNA and the secretion of IL-1?in the cell supernatant were detected by RT-qPCR and ELISA kit,respectively.It was found that CuO NPs could promote the up-regulation of IL-1?expression and the increase of IL-1?secretion in a concentration dependent manner,which confirmed the ability of CuO NPs to induce the inflammatory response of J774A.1 macrophage.Finally,the caspase-1 specific inhibitor Z-YVAD-FMK or transfection of small interfering RNA fragments to down-regulate the expression of NLRP3 protein could significantly inhibit caspase-1 p20 protein expression,caspase-1 activity and IL-1?release,suggesting that NLRP3 inflammasome plays a key role in the inflammatory response induced by CuO NPs.Part ?:This part aims to study the specific molecular mechanism of CuO NPs-induced activation of NLRP3 inflammasomes in J774A.1 macrophage.Firstly,the organelle localization of J774A.1 macrophage after phagocytosis of CuO NPs was observed by TEM,and it was found that CuO NPs were phagocyted by J774A.1macrophage and transferred to lysosome.The effect of CuO NPs on lysosome function was further investigated by using the fluorescent probe and cathepsin B substrate.Confocal microscope observation showed that CuO NPs could induce lysosome damage and abnormal function as well as the release of cathepsin B.The cathepsin B specific inhibitor CA-074 Me could partially alleviate the activation of caspase-1 and the maturation and secretion of IL-1?induced by CuO NPs,and the role of lysosomal damage and cathepsin B in CuO NPs-induced NLRP3 inflammasome activation was identified.On the other hand,due to the strong solubility of CuO NPs under acidic conditions,the solubility of CuO NPs in cell lysosomes was further investigated.The solubility of CuO NPs in cells was determined by ICP-OES.It was found that CuO NPs could release a large amount of Cu²⁺in J774A.1 macrophage.EPR was used to detect the ability of CuO NPs and Cu²⁺to generate ROS.EDTA can completely eliminate the ROS generated by CuO NPs and Cu²⁺.The ROS fluorescent probe DCFH-DA was further used to investigate the ability of CuO NPs to induce oxidative stress in J774A.1marophage,and the results showed that CuO NPs increased ROS production in cells and leaded to oxidative stress.Copper chelating agent TTM inhibited the intracellular oxidative stress and NLRP3 actiation induced by CuO NPs.Finally,ROS scavenger NAC inhibited CuO NPs-induced caspase-1 activation and IL-1?release,indicating that Cu²⁺dissolved by CuO NPs was the key to cause cellular oxidative stress,which further mediated the activation of NLRP3 inflammasomes.In addition,it was found that the exposure of CuO NPs induced the activation of TLR4/MyD88/NF-?B signaling pathway in J774A.1 macrophage to accumulate pro-IL-1?.