Effects Of Propofol On Eryptosis Of Human Erythrocytes Induced By Hydrogen Peroxide In Vitro

Objective Erythrocytes in vivo and in vitro stimulated by environmental factors would appear the phenomenon similar to anoptosis, characterized by cell membrane phosphatidylserine (PS) exposed, volume decreased, cell membrane vesiculated. In order to distinguish from apoptosis, Lang defined it eryptosis or suicidal erythrocyte death. Exposing phosphatidylserine are recognized, bound, engulfed, and degraded by macrophages, this procession could avoid hemolysis caused by the release of hemoglobin tissue and organ damage, but erythrocytes shortened life expectancy, in severe cases it can cause anemia. Will hydrogen peroxide (H2O2) cause eryptosis? What is the metabolism of erythrocytes of nitric oxide (NO), nitrite (NO2-) and nitrate (NO3-)? What is the effect of propofol (PPF) on eryptosis as well as NO, NO2-and NO3-metabolism? In order to explain these issues, this experimental were designed to:①build up the erytosis model in vitro induced by H2O2;②nvestigate whether or not propofol (PPF) protect erythrocytes induced by H2O2;③detection of intracellular NO and NO?-and NO3-content changes;④investigate the protective effect of propofol on human erythrocytes and antioxidant mechanisms of injury.Methods The experiments were divided into two parts. The first part:build up the erytosis model in vitro induced by H2O2. Erythrocytes from whole blood of healthy volunteers were prepared in2%erythrocyte suspension, the suspension containing (mmol/1):NaCl145, KCl4, CaCl22, glucose5, pH=7.4. Erythrocyte suspension was divided into five groups (n=3):blank control group (C), H2O250μm/l group (H50), H2O2100μm/l group (H100), H2O2200μm/l group (H200) and H2O2400μm/l group (H400). Groups were set into the constant temperature shock incubation at50rpm,37°C for1hour. In this study, PS-exposure (annexin Ⅴ binding) and cell volume (forward scatter) were determined by FACS.As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at405nm. The absorption of the supernatant of erythrocyte in distilled water was defined as100%hemolysis. The second part: The effect of PPF pretreatment on H2O2-induced erytosis remodeling in human and mechanism was also studied preliminarily. Erythrocyte suspension was divided into six groups (n=4):①control group (C);②The H2O2group (H);③propofol10μmol/L+H2O2group (P12.5+H);④propofol25μmol/L+H2O2group (P25+H);⑤propofol50μmol/L of+H2O2group (P50+H);⑥propofol100μmol/L+H2O2group (P12.5+H). The time of propofol pretreatment was30min, then stimulated by H2O2200μmol/L. At the same time, group Iono was demonstrated for positive control of calcium ions influx, took group introlipid50μM (In50), group introlipid50μM+H2O2(In50+H) as PPF negative control, took group vitamin E50μM (E50), group vitamin E50uM+H2O2(E50+H) as control group of free radicals removed by antioxidants. Each group PPF pretreatment time was30minutes; they were placed in the incubator of constant temperature shocks at50rpm,37℃for1hour. In this study, cytosolic Ca2+activity (Fluo-4fluorescence), PS-exposure (annexin Ⅴ binding) and cell volume (forward scatter) were determined by FACS. Experimental groups using the Griess method were commissioned by the Beijing Huaying Biotechnology Laboratory detection of NO, NO2-and NO3-content in erythrocyte.Results Part Ⅰ: Compared with group C, H400erythrocyte hemolysis rate was significantly increased (P<0.05vs control group), H50, H100, H200, three groups had no significant hemolysis (P>0.05). The rate of phosphatidylserine exposure of H200and H400groups increased significantly (P<0.05). FSC of H200group compared with group C decreased significantly (P<0.05). Therefore, we choice reaction concentration H2O2200μm for the eryptosis model. Part Ⅱ: The rate of phosphatidylserine exposure: H(15.20±1.01) was lower than P12.5+H (9.55±2.56), P25+H(4.35±3.62), P50+H(3.09±1.66) and P100+H(1.68±0.28).P25+H, P50+H and P100+H compared with group H, the difference was statistically significant (P<0.05). FSC values: compared with the H group (1767.67±9.19), FSC of P12.5+H, P25+H, P50+H and P10+H groups were increased, they were1783.83±33.26,1797.21±97.13,1810.61±16.28and1792.97±24.83, respectively, of which P50+H group compared to group H, the difference was statistically significant (P<0.05). Ca2+concentration:Compared with group C, H group Ca2+fluorescence intensity was no significant change (P>0.05). Compared with group H, the PPF pretreatment concentrations of Ca2+fluorescence intensity increased, but the difference was no statistically significant (P>0.05). Content of NO:H group (2.40±0.02) was lower than group C (2.99±0.05)(P<0.05), P25+H (3.03±0.13) and P50+H (3.14±0.11) were dose-dependent significantly improved (P<0.05).NO2-content:the H group (22.54±0.14) was lower than group C (24.01±0.33)(P<0.05),and P12.5+H group (22.93±0.16), P25H group (24.05±0.71) P100+H group (27.61±2.99) were higher than the H group (P<0.05). NO3-content:H group (44.77±4.02) and other PPF concentration groups all were higer than in group C (31.14±1.48)(P>0.05).Conclusion (1) A small doses of H2O2in vitro induces eryptosis, characterized by PS exposed, cell shrinkage, but the concentration of intracellular calcium did not change significantly;(2) propofol clear free radicals in protection of erythrocyte resistance to oxidative damage;(3) eryptosis induced by H2O2,this has nothing to do with of the Ca2+ flux, propofol has no effect on the Ca+flux;(4) NO and NO2-content in erythrocyte induced by H2O2decreased, while NO3-increased. Clinical dose of PPF can improve the oxidative damage of erythrocyte in the NO and NO2-content, while it did not inhibit the rise of NO3-,what is more, it increased NO3-content in erythrocyte induced by H2O2;(5) The reaction concentration of H2O2200μm/1makes "nitrate-nitrite-nitric oxide pathway" erythrocyte to the oxide phase change, the PPF inhibit this change.