Hepatocyte-specific TMEM16A Deficiency Alleviates Hepatic Ischemia/Reperfusion Injury Via Suppressing GPX4-medited Ferroptosis

Objective:Ischemia/reperfusion（I/R）-induced liver injury with severe cell death is a major complication of liver transplantation.Ferroptosis is programmed cell death characterized by lipid peroxidation.A growing number of studies have reported that ferroptosis is involved in hepatic I/R injury.Transmembrane member 16A（TMEM16A）,a component of hepatocyte Ca²⁺-activated chloride channel,has been implicated in a variety of liver diseases.Many studies show that TMEM16A can cause cumulation of ROS which is trigger cell membrane injury by lipid peroxidation.However,whether TMEM16A affects hepatic I/R injury by regulating ferroptosis remains unknown.Methods:To learn about the function of TMEM16A in hepatocyte,hepatocyte-specific TMEM16A knockout mice,AAV-mediated hepatocyte-specific overexpression of TMEM16A mice and their corresponding littermate controls were performed in a partial hepatic I/R operation which is ischemia for 90 min and reperfusion to 0–24 h to examine the effect of TMEM16A on hepatic I/R injury.Mechanistically,corresponding plasmids had been transfected in 293T cell line and LO2 cell line to observe protein interaction of TMEM16A and glutathione peroxidase 4（GPX4）by CO-IP tests.Subsequently,we established the AAV which is lack of the TMEM16A–GPX4 interaction domain and injected into mice to see if the interaction of TMEM16A and GPX4 is important for liver I/R injury.Results:（1）TMEM16A expression increased in liver samples from patients and mice with I/R injury,which was correlated with liver damage progression.（2）Hepatocyte-specific TMEM16A knockout alleviated I/R-induced liver damage in mice,ameliorating inflammation and ferroptotic cell death.However,mice with hepatic TMEM16A overexpression showed the opposite phenotype.（3）The CO-IP tests show that TMEM16A interacted with GPX4 to induce its ubiquitination and degradation,thereby enhancing ferroptosis.（4）Disruption of TMEM16A–GPX4 interaction abrogated the effects of TMEM16A on GPX4 ubiquitination,ferroptosis,and hepatic I/R injury.Conclusion:Our results demonstrate that TMEM16A exacerbates hepatic I/R injury by promoting GPX4-dependent ferroptosis.TMEM16A–GPX4 interaction and GPX4ubiquitination are therefore indispensable for TMEM16A-regulated hepatic I/R injury,suggesting that blockades of TMEM16A–GPX4 interaction or TMEM16A inhibition in hepatocytes may represent promising therapeutic strategies for acute liver injury.