Hepatotoxicity Of CdSe/ZnS Quantum Dot Nanoparticles And The Underlying Molecular Mechanism

BackgroundQuantum dots(QDs) are semiconductor nanocrystals which are increasingly applied in biological and medical fields due to their superior photophysical properties, small size and modifiable surface. However, the in vitro and in vivo toxicity has been an important issue in biological and medical applications of QDs. Liver is the major site of its biodistribution and accumulation, and although many evidence regarding hepatotoxicity of QDs, the underlying mechanism is obscure. NLRP3(NLR pyrin domain containing 3) inflammasome is a recently discovered protein complex which can be activated by various pathogens, molecules and environmental irritants. Activated NLRP3 inflammasome will result in active caspase-1 which then leads to IL-1β and IL-18 mature and also induces a proinflammatory form of cell death named pyroptosis. Aberrant NLRP3 inflammasome activation can result in liver inflammation, dysfunction and fibrosis. Thus, this study investigates the role of NLRP3 inflammasome activation in QDs-induced hepatotoxicity, and the roles of mitochondrial reactive oxygen species(mtROS) and Ca2+ signals in QDs-induced NLRP3 inflammasome activation.Methods(1) Based on the characterization of the Cd Se/Zn S QDs, the cellular uptake and cytotoxicity of QDs defined by cell viability and LDH release were detected in L02 hepatic cells. Then, the NLRP3 inflammasome activation was determined by western blot after QDs exposure. And the pyroptosis were detected by flow cytometry after treatment with QDs. The role of NLRP3 inflammasome was investigated by siRNA against NLRP3 and ASC.(2) The C57BL/6 mice were administrated with 10 nM/kg QDs via tail vein injection for 14 days. The histological examination of liver was performed by hematoxylin and eosin(H&E) staining. The mRNA expressions of TNF-α, IL-1β, IL-6, CXCL1 and MCP-1 were determined by real-time PCR. Neutrophil infiltration was assessed by MPO levels by western blot. And the liver function was evaluated by serum levels of ALT, AST, ALP and γGT. The activation of NLRP3 inflammasome was also detected in the liver after QDs administration. And the role of NLRP3 inflammasome in QDs-induced liver damage was investigated using a NLRP3 knockout mouse strain.(3) The role of mt ROS in QDs-induced NLRP3 inflammasome activation and pyroptosis in hepatocytes was investigated using the ROS scavenger NAC and mt ROS scavenger Mito-TEMPO. And by NAC and Mito-TEMPO administration in mice, the role of mt ROS in NLRP3 activation and subsequent inflammation and dysfunction in liver was evaluated. And the role of Ca2+ in QDs-induced NLRP3 activation and pyroptosis in hepatocytes was investigated using Ca2+ inhibitors of 2-APB and BAPTA-AM.Results(1) Typical physicochemical characteristics were observed in the CdSe/ZnS QDs used in this study. After uptake by hepatocytes, QDs induced dose-dependent decrease in cell viability and increase in LDH release. QDs exposure activated NLRP3 inflammasome and pyroptosis in hepatocytes, but siRNA against NLRP3 and ASC reduced QDs-induced hepatocyte pyroptosis.(2) QDs administration induced inflammatory foci, and increased m RNA levels of TNF-α, IL-1β, IL-6, CXCL1 and MCP-1, elevated MPO protein expression in liver. QDs also caused liver dysfunction. QDs resulted in NLRP3 inflammasome activation in the liver, which was demonstrated to mediate the QDs-induced liver inflammation and dysfunction by using a NLRP3 knockout mouse train.(3) NAC and Mito-TEMPO inhibited QDs-induced NLRP3 activation and pyroptosi in hepatocytes. Also, NAC and Mito-TEMPO suppressed liver inflammation and dysfunction after QDs exposure. 2-APB and BAPTA-AM treatments reduced QDs-induced mtROS generation, NLRP3 inflammasome activation and pyroptosis in hepatocytes.ConclusionThe activation of NLRP3 inflammasome and subsequent pyroptosis is the novel mechanism underlying QDs-induced hepatotoxicity. QDs induced liver inflammation and dysfunction via NLRP3 activation and pyroptosis in hepatocytes. The QDs-induced Ca2+ mobilization initiated this progress: QDs increase cytoplasmic Ca2+, which then results in mt ROS production. Then, elevated cytoplasmic Ca2+ and mt ROS cause NLRP3 activation, leading to hepatocyte pyroptosis, liver inflammation and dysfunction. This study reveals a novel mechanism for the hepatotoxicity of QDs, and facilitates control of the side effects of QDs application.