Mechanistic insights into aniline-induced liver injury: Role of the mmu_circ_26984/Myh9/NLRP3 axis and modulation by N-acetylcysteine.

ABSTRACT

Aniline is a widely used chemical. Chronic or high-dose exposure to aniline can lead to hepatocellular damage. Although the hepatic pathogenicity of aniline has been established in previous studies, studies involving pathogenic genes during aniline-induced liver injury are limited. Our study first discovered and identified the role and mechanism underlying a new circRNA mmu_circ_26984 in aniline-induced chemical liver injury. Further, we discuss the protective effect of N-acetylcysteine (NAC) in this pathway. After constructing in vitro and in vivo models of aniline treatment, we screened the circRNA with significant differences in expression in AML12 cells from control and aniline-treated groups by circRNA microarray analysis. Next, using RNA pulldown, liquid chromatography-mass spectrometry (LC-MS), and RNA immunoprecipitation, we analyzed the relationship between mmu_circ_26984 and myosin heavy chain 9 (Myh9). Subsequently, we determined the specific mechanism of action of mmu_circ_26984 and Myh9 in aniline-induced liver injury and the protective effect of NAC against aniline-induced liver injury process using Cell Counting Kit-8, Western blot, RNA extraction, a reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence in situ hybridization, immunohistochemistry, and immunofluorescence. The expression of mmu_circ_26984 was significantly increased in liver tissues and AML12 cells of aniline-treated mice compared with the control group. This high expression of mmu_circ_26984 increased the expression of injury-related inflammatory factors, such as NLRP3, Caspase-1, IL-18, and IL-1ß in vivo and ex vivo, which exacerbated the level of liver injury. The interaction of mmu_circ_26984 with Myh9 also affected the course of liver injury. Mmu_circ_26984 overexpression and reduced treatment affected the levels of Myh9 expression in AML12 cells, as well as downstream inflammatory factors associated with injury, such as NLRP3. In addition, NAC reduced the process of liver injury mediated by the mmu_circ_26984/Myh9/NLRP3 axis. In conclusion, mmu_circ_26984 is a potential molecular marker and therapeutic target in the process of aniline-induced liver injury that can mediate aniline-exposure-induced liver injury via modulation of the mmu_circ_26984/Myh9/NLRP3 axis, and NAC can effectively attenuate the effect of this liver injury.