Apoptosis as an underlying mechanism in lymphocytes induced by riboflavin and ultraviolet light

Riboflavin plus UV light pathogen reduction technology (RF-PRT) is an effective method for inactivating donor-derived leukocytes (DDLs) in blood components. Literature data have shown that reactive oxygen species (ROS) increased in lymphocytes after RF-PRT treatment. Sustained high levels of ROS may abolish the endogenous antioxidant system, leading to damage to proteins, lipids, and nucleic acids, resulting in cell apoptosis. Nevertheless, whether riboflavin plus UV light can trigger leukocyte apoptosis remains obscure. In this study, a pool-and-split design, ABO/D-matched lymphocytes treated with RF-PRT or UV light or left untreated. After treatment, the level of ROS and intracellular calcium were measured in samples. Changes in the protein expression of cleaved PARP, Bax, and Bcl-2 and the activities of caspase-3 and caspase-9 were determined by immunoblot analysis or luminometer, respectively. Cell apoptosis was evaluated by flow cytometry. The effect of ROS on apoptosis was assessed. The RF-PRT treatment significantly augmented ROS production, intracellular calcium concentration. The pro-apoptotic proteins expression levels of Bax, but did not the anti-apoptotic protein Bcl-2, were markedly increased after the RF-PRT treatment. Furthermore, the percentage of apoptotic cells was increased in RF-PRT-treated lymphocytes compared to UV-treated cells or untreated cells. Moreover, the inhibition of ROS generation partially neutralized the apoptosis effects of riboflavin plus UV treatment. These findings revealed that RF-PRT-treated lymphocytes significantly increase the proportion of apoptotic cells by promoting ROS generation delineation of the biochemical processes influenced by RF-PRT are a necessary step to provide novel insights into the riboflavin pathogen inactivation technology.