Hemolysis-associated release of hemoglobin induces mitochondrial oxidative phosphorylation (OXPHOS) disturbance and aggravates cell oxidative damage in grass carp (Ctenopharyngodon idella)

Abstract

The liver is a key site for the removal of cell-free hemin during hemolysis. However, the mechanism underlying liver damage caused by hemolysis in teleost hemolytic disorderss remains unclear. In this study, the hemin incubation of grass carp liver cells (L8824) and phenylhydrazine (PHZ) injection were employed to simulate in vitro and in vivo hemolysis models. The Cell Counting Kit (CCK) assay results of the L8824 cells showed that the hemin caused obvious cell death and exhibited concentration-dependent characteristics. Furthermore, hemin stimulation significantly increased intracellular iron content, markedly enhanced intracellular ROS (reactive oxygen species) production, triggered the activation of genes linked to iron metabolism, and disrupted mitochondrial structural integrity. The quantitative real-time PCR (qRT-PCR) assay and enzyme activity findings indicated that the hemoglobin (Hb) treatment activated the activity and expression of mitochondrial respiratory chain complexes, while the addition of compound inhibitors I, II, and III could rescue hemin-induced cell death. Finally, a hemolysis model was established via intraperitoneal injection of PHZ in the grass carp. Histopathological analysis and in vivo transcriptome data showed that PHZ-induced hemolysis resulted in liver inflammation and iron and collagen fiber buildup. Additionally, immunofluorescence and immunohistochemical data indicated it enhanced the ROS generation, malondialdehyde (MDA), and 4-hydroxy-2-nonenal (4-HNE), destroyed the mitochondria, and up-regulated the transcription of mitochondrial respiratory chain complexes. In summary, the cell-free Hb released during hemolysis increased iron deposition, disrupted iron metabolism homeostasis, and caused oxidative stress. Consequently, this destroyed mitochondria function and ultimately exacerbated cell death.