Modulation Of The Anti-Oxidizing Ability And Nitric Oxide Metabolism Of Human Erythrocytes By Propofol

Objective: The clinical observation of propofol （PPF） that protect erythrocytes againstoxidative damage has been reported. We found that PPF could inhibit hydrogenperoxide-induced erythrocytes apoptosis. In recent years, many studies showed that PPFmodulate the nitric oxide （NO） on endothelial cells and neutrophils. Propofol wouldhave a negative impact on microcirculation. While the modulation of PPF on theantioxidant system of erythrocyte haven’t been reported. Recently, some studies haveshown that erythrocytes express a functional endothelial nitric oxide synthase （eNOS）that can potentially serve as an intraluminal NO source. The integrity of function andstructure of erythrocytes are easily affected by the change of endogenous andexogenous active oxygen content. Erythrocytes was a reliable model for research.Weuse this model from antioxidant enzyme system and metabolism of nitric oxide twoaspects of in vitro and in vivo clinical observations, reveal the mechanism of PPF effecton human rerythrocytes function.Methods:1. In vitro: Red blood cells pretreatment with different concentrations ofpropofol.Then induce red blood cells generated oxidative stress. Experimental sampleswere divided into10groups: control group （C group）, H₂O₂group （H group）, propofol12.5μmol/L group（P12.5）, propofol25μmol/L group（P25）, propofol50μmol/Lgroup（P50）, propofol100μmol/L group（P100）, propofol12.5mol/L+H₂O₂（P12.5+H group）, propofol25μmol/L+H₂O₂（P25+H group）, of propofol50mmol/L+H₂O₂（P50+H group） and propofol100mol/L+H₂O₂group （P100+H group）. After all sample were processed, incubated for3h, then detected the levels of CAT, GSH,MDA, GSSG,GSH-PX and NO, eNOS, NO₃^-NO₂^-in erythrocytes.2. In vivo:18consecutive patients were chosen from those scheduled for elective surgery with generalanesthesia. All patients were randomly divided into two groups: intravenous anesthesiaand inhalation anesthesia. The intravenous anesthesia group use PPF to induce andmaintain anesthesia.The inhalation anesthesia group use sevoflurane to induce andmaintain anesthesia. Two blood samples were obtained: The pre-anesthesia sample wasobtained with the patient in decubitus on the operating table;90min after PPF infusionor sevoflurane inhalation from a vein in arm contralateral to the cannulated arm.Detected the levels of CAT, GSH, MDA, GSSG, GSH-PX and NO, eNOS, NO₃^-, NO₂^-in erythrocytes and serum.Results: Part I: the erythrocyte antioxidant index changes:（a） In vitro experiments:（1）the rate of the hemolysis: C group （11.42%±2.53%） compared with the H group （14.28%±3.12%）, hemolysis rate was no significant difference. P50group （23.8%±4.07%） andP100group （30.2%±5.04%） the hemolysis rate increased significantly （P <0.05）;compared with the H group, P12.5group （12.3%±3.12%） and P25+H group （13.9%±2.08%） hemolysis rate decreased, while P100+H group （37.4%±3.0%） the hemolysisrate increased （P <0.05）.2.SOD: Compared with the C group （16.55±0.02）, P12.5group （16.68±0.02）, P25group （16.69±0.06）, P50group （16.75±0.05） elevated levelof SOD; H group （16.43±0.04） SOD levels decreased.3.CAT: P50+H group （5.93±0.05） compared with the H group （5.85±0.08）, CAT water increased （P <0.05）; P50group （5.91±0.13） and C group （5.85±0.06） compared to CAT levels （P <0.05）.4.GSH: P50group （9.32±0.17） compared with the C group （8.22±0.15）, GSH levelwas significantly higher （P <0.05）; P25+H （8.84±0.19） group and P50+H （8.91±0.25） group and H group （8.23±0.29） compared to GSH levels （P <0.05）.5.GSH-PX: Compared with the C group （0.47±0.08）, P50（0.69±0.11） and P100（0.82±0.13）（P<0.05） elevated GSH-PX level.6.GSSG: Compared with the C group （0.24±0.03）, H（0.31±0.01） GSSG level increased （P <0.05）.（B） in vivo: CAT level before induction（5.47±0.13）, after intubation （5.52±0.13）, after the induction of30min （5.57±0.16）gradually increased;2h （5.40±0.29） CAT levels decreased after induction. MDA levelsbefore induction （4.63±0.04）, after intubation （4.48±0.01）, after the induction of30min （4.30±0.07）,2h after induction （3.97±0.08） gradually reduce the four time points.SOD levels before induction （15.50±0.04）, after intubation （15.52±0.05）,30min afterinduction （15.56±0.05） hours after induction （15.58±0.05）, four time points graduallyincreased. GSH levels before induction （7.80±0.33）, after intubation （8.05±0.52）,after the induction of30min （8.29±0.6） hours after induction （8.53±0.65） four timepoints gradually increased. GSH-PX levels after intubation before induction （2.22±0.09）,（2.32±0.13）, after the induction of30min （2.41±0.15） hours after induction（2.53±0.23） gradually increased four time points. GSSG level before induction （0.29±0.02）, after intubation （0.30±0.02）, after the induction of30min （0.31±0.03）, after theinduction of2h （0.32±0.03） gradually increased four time points.Compared to the second part: NO metabolic indicators:（a） in vitro:1.NO: C group（4.49±0.14）, P12.5group （3.44±0.55）, P25group （3.73±0.49） and P100group （3.63±0.51） red blood NO levels decreased （P <0.05）; P50group （4.72±0.58） red blood NOlevels higher than the rest of the group; compared with C group （4.49±0.14）, H （4.96±0.52） NO level increased （P <0.05）. Compared with the H group, P12.5+H group （3.78±0.54）, P25+H group （4.26±0.68）, P50+H group （3.99±0.45）, P100+H group（4.87±0.47） four groups of red blood cells NO levels decreased （P <0.05）.2.eNOS:Compared with the C group （2.09±0.41）, H （2.33±0.13） elevated erythrocyte eNOSlevels; the P12.5group （1.61±0.40）, P25group （1.75±0.48） and the P100H group（1.68±0.39） erythrocyte eNOS levels decreased （P <0.05）.3. NO₃^-: C group （50.06± 1.62） compared to the H group （45.78±2.13） red blood cell NO₃^-level reduced;compared with the H group, P12.5+H group （56.83±2.53）, P25+H group （59.62±1.06）, P50+H group （53.25±2.77） NO₃^-level （P <0.05）. NO₂^-: with the C group（18.66±1.48） compared to the H group （13.32±2.04） red blood cells NO₂^-level lower（P <0.05）; compared with the H group, P12.5group （21.47±2.53） red blood cellsNO₂^-levels （P <0.05）.（B） in vivo:1.NO: NO level at the beginning of the inductiongradually reduced. NO levels in the hours after anesthesia （3.90±0.06） than beforeinduction （4.60±0.04）, after intubation （4.45±0.09）, after the induction of30min（4.42±0.09） were significantly lower （P <0.05）.2.eNOS: before induction （4.60±0.04）, after intubation （4.45±0.09）, after the induction of30min （4.42±0.09） hoursafter anesthesia （3.90±0.06） at each time point eNOS levels decreased.3.iNOS: before induction （2.66±0.04）, after intubation （2.64±0.06）,30min （2.35±0.03） after induction of anesthesia after2h （2.75±0.02） at each time point iNOS weregradually reduced.4.NO₃^-: before induction （68.21±2.95）, after intubation （66.88±4.09）, after the induction of30min （58.36±1.84）, after anesthesia2h （53.84±2.74） ateach time point NO₃^-levels decreased （Figure4）.5.NO₂^-: before induction （17.52±0.71）, after intubation （16.49±0.75）,30min after induction （15.47±0.47）, afteranesthesia2h （14.24±0.43） at each time point NO₃^-level gradually decreasedConclusion:（1） Propofol enhanced erythrocytes antioxidant capacity by increased thelevels of SOD, CAT, GSH and GSH-PX;（2） There were two isform of NOS in erythrocyte, which were eNOS andiNOS; The level of iNOS was higher than eNOS;（3） Propofol could inhibit eNOS and iNOS activity in erythrocytes,disgraded the levels of NO in human erythrocyte; （4） H₂O₂could enhance eNOS activity, upgraded the levels of eNOS,thelevel of NO in human erythrocytes were increased.