Chloantraniliprole(CAP)是一种双酰胺农药,主要用于农业生产活动和稻鱼共培养系统中的虫害防治。CAP残留会导致非靶标生物淡水鱼的肝损伤。然而,目前尚不清楚CAP暴露引起的鱼类肝损伤是否与线粒体功能障碍介导的线粒体自噬有关,铁性凋亡,和细胞因子。因此,我们建立了草鱼肝细胞模型暴露于体外不同浓度的CAP(20、40和80μM)。MitoSOX探针,JC-1染色,免疫荧光双重染色,Fe2+染色,脂质过氧化染色,qRT-PCR,采用Westernblot方法验证CAP诱导肝损伤的生理调控机制。在本研究中,CAP处理组表现出抗氧化剂相关酶活性的下调和过氧化物的积累。CAP处理诱导草鱼肝细胞中线粒体活性氧(mtROS)水平的增加和线粒体裂变/融合(Drp1,Fis1,Mfn1,Mfn2和Opa1)基因的表达改变。此外,线粒体自噬(Parkin,Pink1,p62,LC3II/I,和Beclin-1),铁性(GPX4,COX2,ACSL4,FTH,和NCOA4),和细胞因子(IFN-γ,IL-18、IL-17、IL-6、IL-10、IL-1β、IL-2和TNF-α)相关基因表达显著改变。总的来说,这些发现表明,CAP暴露驱动线粒体自噬激活,铁性凋亡的发生,通过触发mtROS-线粒体裂变/融合轴介导的线粒体功能障碍,以及草鱼肝细胞中细胞因子稳态失衡。本研究从生理生化角度部分解释了杀虫剂CAP诱导草鱼肝细胞损伤的生理调控机制,为评价CAP环境残留对非靶标生物的安全性提供了依据。
Chlorantraniliprole (CAP) is a bis-amide pesticide used for pest control mainly in agricultural production activities and rice-fish co-culture systems. CAP residues cause liver damage in non-target organism freshwater fish. However, it is unclear whether CAP-exposure-induced liver injury in fish is associated with mitochondrial dysfunction-mediated mitophagy, ferroptosis, and cytokines. Therefore, we established grass carp hepatocyte models exposed to different concentrations of CAP (20, 40, and 80 μM) in vitro. MitoSOX probe, JC-1 staining, immunofluorescence double staining, Fe2+ staining, lipid peroxidation staining, qRT-PCR, and Western blot were used to verify the physiological regulatory mechanism of CAP induced liver injury. In the present study, the CAP-treated groups exhibited down-regulation of antioxidant-related enzyme activities and accumulation of peroxides. CAP treatment induced an increase in mitochondrial reactive oxygen species (mtROS) levels and altered expression of mitochondrial fission/fusion (Drp1, Fis1, Mfn1, Mfn2, and Opa1) genes in grass carp hepatocytes. In addition, mitophagy (Parkin, Pink1, p62, LC3II/I, and Beclin-1), ferroptosis (GPX4, COX2, ACSL4, FTH, and NCOA4), and cytokine (IFN-γ, IL-18, IL-17, IL-6, IL-10, IL-1β, IL-2, and TNF-α)-related gene expression was significantly altered. Collectively, these findings suggest that CAP exposure drives mitophagy activation, ferroptosis occurrence, and cytokine homeostasis imbalance in grass carp hepatocytes by triggering mitochondrial dysfunction mediated by the mtROS-mitochondrial fission/fusion axis. This study partly explained the physiological regulation mechanism of grass carp hepatocyte injury induced by insecticide CAP from the physiological and biochemical point of view and provided a basis for evaluating the safety of CAP environmental residues to non-target organisms.