The Protection Mechanism Of P2X7 Receptor Blocker In The Injury Of Erythrocyte Induced By Oxidative Damage

BackgroundOxidative stress plays an important role in many physiological and pathological phenomena.RBC,one of the most important cell in the maintain of life,have been used as a model to study the oxidative damage of biofilm because its high degree of vulnerability to oxidative stress.P2X7 receptor is a purinergic receptors which can respond to the extracellular nucleotides.The activation of P2X7 receptor can lead to the rapid flow of non-selective cation,eventually causing swelling and rupture of cells,namely hemolysis.Therefore,researching the mechanism of P2X7 receptor in the injury of erythrocyte caused by oxidative stress,which can provide new ideas and methods for damage of erythrocyte caused by oxidative stress.Objectives1 To explore the effect of AAPH in erythrocyte through treated with various concentrations of [ 2,2-Azobis（2-methylpropionamidine）dihydrochloride,AAPH].2 To explore the relationship between P2X7 receptor and oxidative injury of erythrocyte caused by oxidative stress through the protective research by BBG.3 To explore the mechanism of P2X7 receptor in the oxidative injury of erythrocyte through measuring the evaluation indicators（hemolysis,osmotic fragility,phosphatidylserine externalization,intracellular Ca2 + concentration）by microplate reader detection technology 、fluorescence microscopy and flow cytometry technique.Methods1 Erythrocytes were taken from normal male Wistar rats and dubbed to 6.25%suspension with PBS solution and divided into six groups.They were treated with variousconcentrations of AAPH（0 mmol / l、10 mmol / l、30 mmol / l、50 mmol / l、80 mmol / l、100 mmol / l）,and incubated at 37 ℃ thermostat water bath.After incubation,samples were taken at different time points（30 min、60 min、90 min、120 min、150 min、180 min）,and measured the absorbance of hemoglobin at 540 nm（that is OD value）by microplate reader.The absorbance of hemoglobin released in the deionized water when completeiy rupture as the basis value to calculate the hemolysis of erythrocytes in the PBS solition.The formula is that :hemolysis（%）=（OD value of the sample in PBS solution-OD value of pure PBS solution）/（OD value of the sample in deionized water-OD value of pure deionized water）.2 An appropriate scathing concentration was chosen to induce the oxidative damage of Erythrocyte according to the time-dependent and concentration-dependent curve caused by AAPH.At the same time,erythrocytes were treated with different concentrations of BBG(（0 umol / l、0.1 umol / l、0.3 umol / l、0.5 umol / l、1 umol / l、3 umol / l、5 umol/ l、10 umol / l）to against oxidative stress.After incubated 1.5 h at 37 ℃,samples were taken and measured the absorbance of hemoglobin in the supernatant at 540 nm by microplate reader.The absorbance of hemoglobin released in the deionized water when completeiy rupture as the basis value to calculate the hemolysis of erythrocytes in the PBS solition.The formula is that :hemolysis（%）=（OD value of the sample in PBS solution-ODvalue of pure PBS solution）/（OD value of sample in deionized water-OD value of pure deionized water）×100%.3 An appropriate protective concentration and a scathing concentration were chosen according to the early results,and explore the protective mechanism of BBG to oxidative damage of erythrocyte induced by AAPH from four aspects,namely,hemolysis,osmotic fragility,phosphatidylserine exposure and the intracellular concentration of Ca²⁺.The hemolysis was expressed by the hemoglobin absorbance at 540 nm.The hemoglobin absorbance in sample supernatant was measured with microplate reader,and the absorbance of hemoglobin released in the deionized water when completeiy rupture as thebasis value to calculate the hemolysis of erythrocytes.As the resistance of erythrocyte in different concentrations of NaCl buffered saline solution（0.3%、0.4%、0.9%,pH = 7.40）was different,by which we can measure the osmotic fragility of erythrocyte.The erythrocytes of phosphatidylserine exposure were labeled with Annexin V-FITC and visually observe in the fluorescence microscopy.The intracellular of Ca²⁺of erythrocytes were labeled with FLUO-3 / AM.We can visually and quantify observe the changes of the Ca2 + concentration in erythrocyte through fluorescence microscopy and flow cytometry technique.Results1 Compared with normal control group（AAPH: 0 mmol/l）,the hemolysis phenomenon appears in the AAPH groups（10 mmol / l、30 mmol / l、50 mmol / l、80 mmol / l、100mmol / l）.At the same time point,the greater of the concentration of AAPH,the larger of the erythrocyte hemolysis;At the same concentration,the longer of action time,the larger of the erythrocyte hemolysis.2 When the concentration of BBG less than 5 umol / l（0.1 umol / l、0.3 umol / l、0.5 umol / l、1 umol / l、3 umol / l）,it can not effectively suppress the erythrocyte hemolysis induced by AAPH.When the concentration of BBG is 5 umol / l or 10 umol / l,the hemolysis rate of erythrocyte induced by AAPH decreased significantly（P <0.05）.3 Compared with the control group,AAPH group: hemolysis rate increased significantly（P<0.01）,osmotic fragility increased significantly（P<0.01）,count of erythrocyte labeled by Annexin V-FITC increased significantly（P<0.01）,Ca²⁺ concentration increased significantly（P <0.01）.After treated with BBG,compared with AAPH group,AAPH + BBG group: hemolysis rate（P <0.05）,osmotic fragility（P <0.05）,the count of erythrocyte labeled by Annexin V-FITC（P<0.05）and Ca2 + concentration of erythrocyte（P<0.05）were reduced significantly.Conclusions1 Oxidative stress can induce the oxidative damage of red blood cells,and thisdamage increases by time-dependently and concentration-dependently.2 P2X7 receptors are involved in the damage of erythrocyte induced by oxidative stress,and P2X7 receptor blocker can effectively inhibit this damage.3 P2X7 receptor blocker can effectively inhibit the oxidative damage of erythrocyte caused by AAPH through inhibiting the P2X7 receptor,reducing the influx of Ca2 +,inhibiting the PS valgus of erythrocyte and ruducing the osmotic fragility.