Role Of NLRP3 Inflammasome In N,N-dimethylformamide-induced Acute Liver Injury In Mice

ObjectiveN,N-dimethylformamide(DMF)is an excellent organic polar solvent that can dissolve a variety of organic substances and is also a well-known hepatotoxicants.Unfortunately,DMF has become one of the common poisons in industrial poisoning in China in recent years.Studies have shown that DMF poisoning is mainly manifested as acute and subacute poisoning,and the pathological phenotype of acute liver injury caused by DMF is characterized by liver parenchymal cell damage,and focal or largescale hepatocyte necrosis accompanied by inflammatory cell infiltration.The activation of inflammasomes plays a crucial role in the inflammatory immune response of the liver.NLRP3 inflammasome plays a major role in the pathogenesis of liver injury caused by various liver toxins such as alcohol and acetaminophen,but whether it participates in the occurrence and progression of DMF-induced liver injury has not been reported.Macrophages are thought to be the main cell type that causes liver damage by inducing activation of NLRP3 inflammasomes,and whether DMF causes liver damage by inducing polarization of hepatic macrophages and activation of NLRP3 inflammasomes has not been studied.In addition,the activation of NLRP3 inflammasome can induce cell pyroptosis,however,whether DMF-induced liver damage is related to the activation of the pyroptosis pathway still needs to be explored.In this study,we intend to investigate explore the mechanism of liver injury caused by acute exposure to DMF and the role of hepatic macrophages in it by focusing on the inflammation-related pathways.MethodsFirstly,the bliss method was used to determine the half-lethal dose(LD50)of DMF to male C57BL/6J mice,and a acute mouse liver injury model induced by DMF was established by gavaging mice with DMF of 1/2 LD50 dose to simulate acute occupational DMF poisoning in humans.Subsequently,GdCl3 was used for the elimination of hepatic macrophages and to investigate the role of NLRP3 inflammasomes and macrophages in DMF-induced acute liver injury by detecting changes in key members of NLRP3 inflammasomes and macrophages in the liver.Finally,HepG2(CYP2E1-HepG2 cells)transfected with human CYP2E1 cDNA was used in the in vitro experiments,the toxicity of DMF to CYP2E1-HepG2 cells was determined by CCK-8 kit,and the time-effect model and dose-response model of DMFinduced CYP2E1-HepG2 cell damage were established to detect cell damage indicators and expression levels of key molecules of NLRP3 inflammasome pathway.Results1、Compared with the control group of mice,the liver coefficient,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities of mice after acute DMF exposure increased significantly.At the same time,the expression of obvious focal large necrosis and inflammatory cell infiltration in the liver of mice gradually increased.2、Acute DMF exposure caused significant increases in serum levels of interleukin 1β(IL-1β),interleukin 6(IL-6),and tumor necrosis factor α(TNF-α)in mice,and was able to cause NLRP3(The nod-,LRR-and pyrin)in the NLRP3 inflammasome pathway in mouse liver domain-containing protein 3,NLRP3),apoptosis-associated speck-like protein(ASC),cysteinyl aspartate specific proteinase1,Caspase-1)and elevated expression of IL-1β proteins and aggregation and M1-type polarization of hepatic macrophages.3、GdCl3 can significantly inhibit the increase of liver coefficient,serum AST and ALT activity in mice caused by acute exposure to DMF,and significantly alleviate liver inflammation and neutrophil infiltration.At the same time,it can significantly reduce the upregulation of protein expression of NLRP3,ASC,Caspase-1 and IL-1βcaused by acute exposure to DMF,as well as the increase of serum IL-1β,TNF-α and IL-6 levels,and inhibit the intrahepatic infiltration of macrophages and the expression of M1-type polarization markers.4、Acute exposure to DMF can lead to an increase in the expression of GSDMD protein in the liver of mice,and GdCl3 can inhibit the increase in DMF-induced GSDMD expression.5、DMF(150 mM)can induce changes in the morphology of CYP2E1-HepG2 cells,cell shrinkage and rupture,blurred edges,and death.The level of lactate dehydrogenase(LDH)in the cell culture medium was increased,but the protein expression of Caspase-1 and GSDMD in the cells did not change significantly.Conclusions1.DMF-induced liver damage in mice is accompanied by the activation of hepatic NLRP3 inflammasomes,while the liver macrophage scavenger GdCl3 can significantly inhibit DMF-induced Activation of NLRP3 inflammasome and liver damage.These results suggest that activation of the macrophage NLRP3 inflammasome pathway may be a key cause of DMF-induced acute liver injury.2.DMF can cause CYP2E1-HepG2 cell death,while the protein expression of Caspase-1 and GSDMD were not altered.These results indicate that DMF may not cause CYP2E1-HepG2 cell death by pyroptosis.