In Vivo Analysis of Necrosis and Ferroptosis in Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease states such as chronic inflammation and fibrosis. In most liver diseases, immunological responses caused by tissue damages or viral infection contribute to the pathological advances, and various types of cell death have been reported to be implicated in their pathogenesis. However, the conventional detection of necrosis in vivo is not currently available, whereas the detection method for apoptosis has been relatively well-established. We recently reported a method for the in vivo detection of necrotic cells in liver disease models by an intravenous injection of Propidium Iodide (PI) into mice. We also provide standard methods for the evaluation of lipid accumulation and fibrosis characteristic of NASH. In addition, by utilizing these procedures and a murine model of steatohepatitis, we showed that ferroptosis, a type of regulated necrotic cell death, could be involved in the pathogenesis of NASH. These approaches allow us to explore the pathophysiological roles of cell death in liver diseases.

Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.