Carbon tetrachloride induced mitochondrial division, respiratory chain damage, abnormal intracellular [H+] and apoptosis are due to the activation of 5-HT degradation system in hepatocytes

Abstract

Previously we found that acute liver injury (ALI) with inflammation caused by carbon tetrachloride (CCl₄) was associated with the activation of the 5-HT degradation system (5DS), which includes monoamine oxidase A (MAO-A), the 5-HT_2A receptor, and 5-HT synthases in hepatocytes. This study aimed to determine the role of 5DS in mitochondrial damage and apoptosis. In hepatocyte LO2 cells, CCl₄ activated 5-HT_2A receptor at the gene level, and then 5-HT_2A receptor mediated the expression of 5-HT synthase and MAO-A at the gene level. Suppression of 5DS with the 5-HT_2A receptor antagonist, MAO-A inhibitor, or gene silencing MAO-A significantly reduced the CCl₄-induced production of mitochondrial reactive oxygen species (ROS). The ROS-associated upregulation of mitochondrial division proteins (FIS1 and DRP1); downregulation of mitochondrial fusion-associated protein 1, respiratory chain proteins (ND1 and CYTB), and ATP6; and decrease of ATP levels were reversed. Moreover, ROS-associated abnormal levels of caspase pathway-associated proteins (Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9) and apoptosis were suppressed. Notably, a combination of 5-HT_2A receptor antagonist and MAO-A inhibitor almost abolished CCl₄ cytotoxicity; abolished mitochondrial membrane potential (MMP) depolarization, mitochondrial structural abnormality, and high mitochondrial pH, with low pH states of the nucleus and cytoplasm. The effects of both were more significant than either alone. LO2 cells exposed to H₂O₂ or depleted mitochondrial ROS showed that ROS induced mitochondrial division and apoptosis and inhibited the levels of respiratory chain proteins. CCl₄-induced abnormalities of ATP generation and MMP were dependent on both ROS and other 5DS-associated factors, probably NH₃. Investigation of CCl₄-induced ALI mice showed that hepatic injury and apoptosis occur at the site of 5DS activation and are significantly inhibited by the 5-HT_2A receptor antagonist and 5-HT synthetic inhibitor in a synergistic manner, as well as mitochondrial damage. Together, we revealed the close relationship between CCl₄-induced activation of 5DS and mitochondrial damage, abnormal intracellular [H⁺], and apoptosis in hepatocytes.

Keywords: 5-HT degradation system; Apoptosis; Intracellular pH value; Mitochondrial division; Mitochondrial respiratory chain; Reactive oxygen species.