The Study On Inhibition Of Suicidal Erythrocyte Death By Indirubin-3’-monoxime

Objective This experimental study aims to explore the potential impact of indirubin-3’-monoxime（IDM）on erythrocytes and its mechanism.Method In this study,the experimental group,control group（solvent group）and blank group were set up with fresh blood samples from 300 healthy blood donors.The experimental groups were IDM-mediated erythrocyte survival rates under different concentration gradients（concentration gradient set to 3μM,6μM,12μM）,and analysis of experimental design group of eryptosis.Erythrocytes were routinely isolated and each group was made into a 0.4%concentration of red cell suspension and incubated for 24 hours.After incubation,Utilizing flow cytometry and confocal laser scanning microscopy,phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate（FITC）.The relative cell volume,expressed in arbitrary units,was evaluated by forward scatter in a flow cytometer.Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca²⁺activity,reactive oxygen species（ROS）formation was assessed by 2,7-dichlorodihydro fluorescein diacetate（DCFH-DA）fluorescence,and ceramide abundance was evaluated by FITC-conjugated specific antibodies.In order to further verify the conclusion,application of ionomycin,tert-butyl hydroperoxide（t-BHP）and lack of energy have the role of promoting erythrocyte apoptosis,increasing calcium influx,decreasing average cell volume,increasing intracellular reactive oxygen species,but these effects were all inhibited by IDM（12μM）.Results IDM（12μM）significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration([Ca²⁺]_i).IDM（3-12μM）did not significantly modify the ceramide level or DCFH-DA fluorescence.Energy depletion（removal of glucose for 24 hours）significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter,and these effects were significantly mitigated by IDM（12μM）.Moreover,the Ca²⁺ionophore ionomycin（1μM,60min）and oxidative stress（30min,0.05mM t-BHP）similarly triggered eryptosis,which was also significantly suppressed by IDM.Conclusions indirubin-3’-monoxime can inhibit the eryptosis and is expected to become a new novel of eryptosis protection agent.This will provide new ideas for the future treatment of erythrocyte apoptosis-induced anemia.