Effects Of Adenoviral-mediated PUMA On Liver Regeneration After Partial Hepatectomy In Mice

PUMA, namely p53 up-regulated modulator of apoptosis, which is a highly conservative pro-apoptotic protein in evolution, was originally discovered as a target of p53. In Bcl-2 family, It belongs to BH3-only proteins sharing only the short BH3 interaction domain, by which they can bind to the pro-survival relatives to induce apoptosis. PUMA, an essential initiator of programmed cell death, can transduct signals of distinct apoptotic stimuli such as oxidative stress, DNA damage, serum deprivation, and hypoxia etc. In physiology state, PUMA is regulated strictly and the protein expression level is relative lower. But in response to a range of stress signals, PUMA is induced in a p53 dependent or independent manner, and shows its potent pro-apoptotic activity.Several members of Bcl-2 family such as Bcl-XL, Bcl-2, and Bax etc. are involved in liver regeneration, but the patterns of these Bcl-2 relatives expression are difficult to explain the phenomenon that there are rarely apoptotic hepatocytes in the early phase. On the other hand, few knows the role of BH3-only proteins in regulating liver regeneration. So we choice PUMA, which has a close relation to the apoptotic pathways that we have known, to explore its role in liver regeneration.Considering these reasons, the main contents we aim to research are as shown in followings. 1. To detect the patterns of PUMA and the other proteins correlated expression in the early phase of liver regeneration. 2. To establish a suitable adenoviral-mediated gene transfer animal model. 3. To examine the effects of PUMA overexpression on liver regeneration after partial hepatectomy in mice.We found in the early phase following partial hepatectomy (0h~72h), PUMA expression was down-regulated transcriptionally, it was related to transcriptional repressor Slug, and independent on p53. The downstream proteins Bcl-XL and Bax were up-regulated gradually, and Bax expression depended on p53. Finally, Caspase 3 was unactivated in the course.To investigate the inner relationship between these proteins, we infused mice with adenovirus vectors carrying PUMA (Ad-PUMA) 48h before partial hepatectomy. Liver regeneration was estimated by hepatocytes proliferation and apoptosis. 48h following infection, 35.7% mice infused with Ad-PUMA at high dose (4×10~8 ifu) died of acute hepatic failure. Mice preteated with Ad-PUMA at low dose (2×10~8 ifu) showed apparent normal, howerver, the mortality within 24h after hepatectomy was 66.7%. Moreover, the livers infected with Ad-PUMA impaired proliferative cells, increased apoptotic ones, delayed response of Bcl-XL, elevated Bax level and showed severe inflammation. So we consider down-regulation of PUMA is useful to liver regeneration. It mayanswer the problem why there are few apoptotic hepatocytes in the early phase. That is, down-regulation of PUMA declines the sensitivity to distinct stress stimuli, elevates the threhold of oxidative stress, and make mass hepatocyte enter into cell cycle successfully. The mechanism may include, 1. PUMA down-regulated by Slug cooperates with Bcl-XL up-regulated to avoid activating Bax. 2. It relieves the inhibition to proliferative hepatocytes , decreases necrosis and inflammation.