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【Objective】 To explore the mechanism of testosterone on eryptosis. 【Methods】 The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups: 2 kinds of eryptosis models induced by hydrogen peroxide (H2O2) in vitro, 3×10-8 mol/L testosterone + 200 μmol/L H2O2 group (group A), 200 μmol/L H2O2 treatment group (group B) and 1×PBS buffer control group (group C). Erythrocytes were collected from 12 well plates (about 2×106/well) at 24 h and 60 h to detect the eryptosis rate, intracellular ROS and [Ca2+ ]i through flow cytometry, and the changes of each index were analyzed by t test. 【Results】 The eryptosis rate(%)at 24 h and 60 h in group A, B and C were 2.61±0.28, 11.25±1.43 vs 7.15±0.95, 28.65±0.74 vs 1.32±0.07, 8.18±0.08 (P<0.01); ROS level(%): 14.52±0.68, 15.26±0.49 vs 16.68±0.60, 21.68±1.10 vs 7.61±0.21, 10.29±1.06 (P<0.01); [Ca2+ ]i(%): 6.54±0.46, 8.93±0.87 vs 11.78±0.76, 14.63±0.80 vs 1.36±0.20, 2.44±0.38 (P<0.01). The eryptosis rate, ROS level and [Ca2 + ]i in group A were significantly lower than those in group B (P<0.01). 【Conclusion】 Testosterone effectively inhibits eryptosis induced by H2O2 in vitro and its mechanism may be related to reducing ROS production and maintaining normal [Ca2+ ]i.
ABSTRACT
AIM:To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H2O2),.and to explore its related mecha-nism. METHODS:The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group,the culture medium was PBS),H2O2group (H group,the culture medium was PBS containing H2O2at final con-centration of 100 μmol/L) and EPO group (E group,the culture medium was PBS containing H2O2at final concentration of 100 μmol/L and EPO at final concentration of 2×104U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion con-centration (Ca2+]i) were also analyzed by flow cytometry. RESULTS:The eryptosis in C group was increased as the in-cubating time extended. The eryptosis in H group was higher than that in C group (P<0.01),while that in E group was lower than that in H group(P<0.01). Meanwhile,ROS production andCa2+]iwere higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION:EPO inhibits eryptosis induced by H2O2and its mechanism may be related to antioxidant effect and change of Ca2+]i.
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[ ABSTRACT] AIM: To investigate whether L-carnitine ( LC) inhibits the eryptosis effect of uremic serum on erythrocytes.METHODS:Erythrocyte suspension (2%) was cultured and divided into 3 groups in vitro: control group ( C group) , uremic serum group ( U group, 30%uremic serum) , and uremic serum+LC group ( L group, 30%uremic serum+200 μmol/L LC) .Erythrocytes were collected at 24 h and 48 h.Eryptosis ( phosphatidylserine expression repre-sents eryptosis) was estimated by flow cytometry with Annexin V staining.The content of reactive oxygen species ( ROS) was also detected.Glutathione ( GSH) was measured by ELISA.RESULTS:Eryptosis in C group was increased as the in-cubating time extended.Eryptosis in U group was higher than that in C group, while that in L group was lower than that in U group.Meanwhile, ROS content was higher and GSH was lower in U group than those in C group.ROS content was low-er and GSH was higher in L group than those in C group.CONCLUSION:LC inhibits uremic serum-induced eryptosis by decreasing ROS and increasing GSH, thus attenuating oxidative stress.
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Objective To investigate the role of tumor necrosis factor-α (TNF-α) on eryptosis.Methods Erythrocytes isolated from mice were put under the treatment of TNF-α at the dose of 1ng/ml for 6,12,24,48 and 72 h,or at different concentrations of 0.1,1 and 10 ng/ml for 24 h.The forward scatter (FSC),phosphatidylserine (PS)exposure and ceramide formation were determined by flow cytometry.Results Compared to control group,the decrease of FSC ((81.5 ± 1.02)％ vs (87.6 ± 0.55)％,P＜0.05),the increasment of membrane PS exposure level and ceramide content ((5.5±1.07)％ vs (2.7±0.17)％,(2.1±0.23)％ vs (0.7±0.26)％,P＜0.01) were observed in erythrocyte under the treatment of TNF-α for 24 h with more obvious tendency over time.Conclusions TNF-α can trigger cell shrinkage,and promote PS exposure and ceramide formation on the membrane of erythrocyte.