Hepatic Ischemia-reperfusion Injury in Mice was Alleviated by Rac1 Inhibition – More Than Just ROS-inhibition

BACKGROUND AND AIMS: Reducing reactive oxygen species (ROS) production has proven an effective way for alleviating oxidative stress during ischemia-reperfusion injury (IRI). Moreover, inhibition of Rac1 could reduce ROS production and prevent oxidative stress injury. Previous studies have suggested a positive interactivation feedback loop between Rac1 and hypoxia-inducible factor (HIF)-1α, the latter being up-regulated early during ischemia. The positive inter-activation between Rac1 and HIF-1α would aggravate ROS production, thereby promoting IRI. This study was designed to verify the effects of Rac1 inhibition on hepatic IRI both at animal and cellular levels and to explore the interaction between Rac1 and HIF-1α during hepatic IRI. METHODS: C57B/6 mice and AML-12 cells were used for the construction of hepatic IRI animal and cell models. Rac1 inhibition was achieved by NSC23766 (a specific Rac1 inhibitor). Lentiviral vectors were used for Rac1 knockdown. At designated time points, serum and liver tissues were collected from the mice and treated cells were collected for further analysis. RESULTS: NSC23766 treatment significantly alleviated the hepatic IRI in mice, manifesting as lower vacuolation score and less apoptosis cells, lower ROS and serum/liver alanine aminotransferase/aspartate aminotransferase levels, and fewer activated inflammatory cells. IRI of AML-12 was also alleviated by 50 µM NSC23766 or Rac1-knockdown, manifesting as reduced cell apoptosis, less extensive interruption of mitochondrial membrane potential, down-regulation of apoptosis, and effects on DNA damage-related proteins. Interestingly, Rac1 knockdown also down-regulated the expression level of HIF-1α. CONCLUSIONS: Our study supports a protective effect of Rac1 inhibition on hepatic IRI. Aside from the classic topics of reducing ROS production and oxidative stress, our study showed an interaction between Rac1 and HIF-1α signaling during hepatic IRI.