Caspase 6 Knockout Protects Mice from IR-Induced Liver Injury

Caspase 6 was knocked out in C57 mice by CRISPR/Cas9 technology (Cyagen Bioscience, Jiangsu, China). All animal experimental protocols were approved by the Animal Ethics Committee of Tianjin First Central Hospital. Liver steatosis was induced in 4-week-old male wild-type (WT) mice or Caspase 6^KO mice fed a high-fat diet (HFD) (Supplementary Table 2 for details) or normal diet (ND) for 12 weeks. Subsequently, mouse liver IR model was established according to our previous study [35 ]. Briefly, the artery/portal vessels were clipped for 90 min and reperfusion was initiated by releasing the clamp. Liver tissue and serum were harvested 6 h after reperfusion. The same procedure was performed in the Sham groups without blocking the blood vessels. Alexa Fluor™ 488-labeled control vectors, Caspase 6-knockdown siRNA, and NR4A1 or SOX9 activation plasmids (2 mg/kg; Santa Cruz Biotechnology) were mixed with mannose-conjugated polymers (Polyplus-transfection, France) at a ratio according to the manufacturer’s instructions. Then they were injected through tail vein of mice 24 h before IR surgeries as described in our previous report [36 (link)]. Mice were then randomly divided into groups without blinding in different settings.