Objective To evaluate the protective effect and underlying mechanism of ulinastatin on hepatic ischemia-reperfusion injury.

Methods Twenty-four male SD rats were divided into three groups: sham operation group (Sham group), hepatic ischemia-reperfusion injury group (HIRI group) and hepatic ischemia-reperfusion injury + ulinastatin pretreatment group (HIRI+UTI group), with 8 rats in each group. The HIRI rat models were established by occluding hepatic portal vein and hepatic artery for 1 h. In the HIRI+UTI group, the rats were intraperitoneally injected with ulinastatin at 30 min before model establishment, and an equivalent amount of normal saline was given in the Sham and HIRI groups. The rats were sacrificed at 6 h after model establishment. Serum samples were collected to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining. Ultrastructural changes of mitochondria in liver tissues were observed by transmission electron microscopy. The expression of glutathione peroxidase 4 (GPX4) was determined by immunofluorescent staining. The contents of malondialdehyde (MDA), glutathione (GSH), Fe, reactive oxygen species (ROS) and GPX4 were detected. The expression levels of GPX4 and acyl-CoA synthetase long-chain family 4 (ACSL4) messenger RNA (mRNA) and proteins in liver tissue were measured.

Results Compared with the Sham group, serum ALT and AST levels were up-regulated, pathological changes such as congestion, hepatocyte necrosis and abnormal hepatic lobule structure were observed, pathological score was increased, the mitochondria shrank, the membrane density was increased, the mitochondrial crest was damaged or even absent, the contents of ROS, MDA and Fe were elevated, the GSH content was decreased, the fluorescent intensity of GPX4 was weakened, the relative expression levels of ACSL4 mRNA and protein were up-regulated, and the relative expression levels of GPX4 mRNA and protein were down-regulated in the HIRI group (all P<0.05). Compared with the HIRI group, serum ALT and AST levels were down-regulated, liver tissue injury was alleviated, pathological score was decreased, mitochondrial shrinkage and crest breakage were mitigated, the contents of ROS, MDA and Fe were down-regulated, the GSH content was up-regulated, the fluorescent intensity of GPX4 was enhanced, the relative expression levels of ACSL4 mRNA and protein were down-regulated, and the relative expression levels of GPX4 mRNA and protein were up-regulated in the HIRI+UTI group (all P<0.05).

Conclusions Ulinastatin may alleviate hepatic ischemia-reperfusion injury in rats probably through inhibiting ferroptosis.