Unconjugated
Oxidative stress induced by ischemia and hypoxia in the livers of donors after brain death (DBD) is associated with poor organ function and low patient survival rates in those receiving DBD liver transplants. Peroxiredoxin 6 (Prdx6) can defend cells against liver damage induced by oxidative stress. The present study aimed to investigate the role of Prdx6 in ischemia‑ and hypoxia‑induced liver damage in DBD livers. Liver tissue samples from ten DBD patients were collected. The control group constituted of six liver samples from patients with liver hemangioma that had accepted tumor excision surgery. Protein expression levels were determined by western blotting, cell viability was assessed using a CCK‑8 assay, intracellular reactive oxygen species (ROS) levels were measured using a ROS assay kit, and phospholipase A2 (PLA2) activity was measured using a PLA2 assay kit. In DBD liver samples, Prdx6 expression was downregulated and the nuclear factor‑κB (NF‑κB) signaling pathway was activated. Furthermore, when human liver L02 cells were exposed to ischemia and hypoxia, the expression of Prdx6 was reduced, causing an increase in reactive oxygen species (ROS); this in turn activated NF‑κB signaling and lowered cell viability (P<0.01). In agreement, overexpression of Prdx6 reduced ROS levels and improved cell viability. It was also demonstrated that inhibition of NF‑κB increased Prdx6 expression, while inhibition of Prdx6 limited PLA2 activity, exacerbating ischemia‑ and hypoxia‑induced cell damage. This data suggests that Prdx6 and its PLA2 activity have a protective role in DBD livers, the expression of which is regulated by NF‑κB. Thus, Prdx6 may be a novel target to alleviate liver damage in DBD.
Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.
