Inhibiting xCT/SLC7A11 induces ferroptosis of myofibroblastic hepatic stellate cells and protects against liver fibrosis

Background and AimsLiver fibrosis develops in the context of excessive oxidative stress, cell death and accumulation of myofibroblasts (MFs) derived from hepatic stellate cells (HSCs). Ferroptosis is a type of regulated cell death that can be caused by inhibiting the cystine/glutamate antiporter xCT. However, while xCT is induced in various liver diseases, its role in HSC activation and liver fibrosis is unknown. We hypothesized that xCT is required for HSCs to antagonize ferroptosis and remain myofibroblastic. MethodsxCT activity was disrupted by siRNA or pharmacological inhibitors in MF-HSC cell lines to determine its effect on redox homeostasis, growth, myofibroblastic activity and viability. xCT expression was then determined by RNA sequencing and RT-PCR during primary HSC activation, and its role in HSC trans-differentiation was assessed. For comparison, xCT expression and function were also determined in primary hepatocytes. Finally, the roles of xCT in HSC accumulation and liver fibrosis were assessed in mice treated acutely with CCl4. ResultsInhibiting xCT in MF-HSCs decreased intracellular glutathione (GSH), suppressed growth and fibrogenesis, and induced cell death. These effects were rescued by antioxidants, an iron chelator, and a canonical ferroptosis inhibitor, but not by inhibitors of apoptosis or necrosis. xCT was dramatically up-regulated during primary HSC activation, and inhibiting xCT suppressed myofibroblastic trans-differentiation and induced ferroptosis. In contrast, healthy hepatocytes were relatively insensitive to ferroptosis induced by xCT inhibition. In vivo, inhibiting xCT systemically reduced MF-HSC accumulation and liver fibrosis after a single dose of CCl4 without exacerbating liver injury or reducing hepatocyte regeneration. ConclusionCompared to healthy hepatocytes, MF-HSCs are exquisitely sensitive to ferroptosis induced by inhibiting xCT. In acutely injured livers, systemic inhibitors of xCT can inhibit fibrosis without worsening liver injury. Further research is needed to determine if this therapeutic window remains sufficiently robust to safely target MF-HSCs and inhibit fibrogenesis in chronically injured liver.