Inhibition of Ca(2+) entry and suicidal erythrocyte death by naringin

Naringin is a dietary flavonoid from citrus fruits with antioxidant and antiapoptotic activity. Similar to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increased cytosolic Ca(2+) activity, e.g. following energy depletion or oxidative stress. The present study thus explored whether naringin interferes with eryptosis. To this end, the cytosolic Ca(2+) concentration was estimated from Fluo3 fluorescence, phosphatidylserine exposure from annexin-V-binding and cell volume from forward scatter in FACS analysis. As a result, energy depletion (48 h glucose removal) and oxidative stress (30 min exposure to 0.3 mM tert-butylhydroperoxide) increased Fluo-3 fluorescence, decreased the erythrocyte forward scatter and enhanced the percentage of annexin-V-binding erythrocytes. Naringin (up to 40 µM) did not significantly modify Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding in the presence of glucose and absence of oxidative stress. Naringin, however, significantly blunted the effect of glucose depletion and oxidative stress on Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding. In conclusion, naringin blunts the increase of cytosolic Ca(2+) concentration, the shrinkage, the cell membrane scrambling and thus the suicidal death of erythrocytes following energy depletion or oxidative stress.