First Exploration Into Protective Effect Of PUMA Inhibitor On The Mouse Hepatic Ischemia-reperfusion Injury

Objective:p53upregulated modulator of apoptosis (PUMA) is a BH-3-only member of Bc1-2family. It’s located in the mitochondrion and remains low expression under normal circumstances. PUMA can be induced by many stimuli, including radiation, virus, chemotherapeutics, et al. PUMA binds Bc1-2-like proteins with its particular BH3domain to inhibit antiapoptosis. PUMA inhibitor mimics these interactions between PUMA and Bcl-2-like proteins so that it could interfere the bindings. In this research we want to test whether PUMA inhibitor could inhibit PUMA expression and exert its protective effect against injuries in two different models.Methods:(1)13.5-day-old ICR mouse babies were sacrificed to get mouse embryo fibroblasts (MEFs).(2) Different concentrations of DOX were used to stimulate MEFs to detect PUMA expression and protective effect of PUMA inhibitor.(3) Hepatic ischemia-reperfusion model was set up with C57/BL6mice and all the experimental mice were divided into three groups, sham group, operation group and PUMA inhibitor group.(4) Serum ALT was tested. PUMA expression at protein and mRNA level was detected. H&E stains and TUNEL were used to judge the degree of injuries together.Results:DOX could induce PUMA expression in MEFs at relatively low concentration, and it decreased if given too much DOX to MEFs.1μM PUMA inhibitor could reduce PUMA expression and it protected MEFs from DOX-induced injury. As for the in vivo model, distinct injuries presented at different ischemic time. PUMA expression increased after the minor ischemia-reperfusion injuries and PUMA inhibitor protected the liver by inhibiting PUMA expression. On the contrary, sever injuries by extending ischemic time caused no differences of PUMA expression between operation group and sham group and PUMA inhibitor showed no protective effect on the liver under such circumstances.Conclusion:Gentle damage activated apoptotic path and up-regulated PUMA expression. PUMA inhibitor mitigated the damage by inhibiting apoptosis initiated by PUMA. However, PUMA was not induced if the injuries were severe for the reason that apoptosis may not be the main path of damages. PUMA inhibitor exhibited its usefulness against certain injuries both in vitro and in vivo and it may be further applied into more fields. PUMAKO mice will be used to test our in vitro results, and PUMA inhibitor will be applied to observe whether it could protect organs during hepatic cold ischemia-reperfusion or transplantation.