Effect Of Nitric Oxide On Oxygen-Carrying Capability Of Erythrocytes And Its Significance

Hypoxia is a common pathophysiological process to ischemia,inflammation,premature birth,cancer,and altitude exposure.Insufficient oxygen supply to the tissue and excessive oxygen demand in the tissue can cause hypoxia.Hypoxia can lead to cell dysfunction,structural damage,tissue damage,and even organ failure.In the absence of oxygen,a series of changes are ignited to adapt to hypoxia.Among them,based on the important role of erythrocytes in oxygen uptake and transport,the change in the quantity and quality of erythrocytes（RBC）plays an important role in hypoxia adaptation.In the high altitude environment,the partial pressure of oxygen decreases as the atmospheric pressure decreases.When the body is exposed to the high altitude,the tendency of oxygen to enter the blood from alveolar is reduced,which result in insufficient oxygen supply to the tissue,and break the balance of supply and demand of oxygen.The body initiates a variety of compensatory mechanisms around oxygen re-equilibration,including increasing the number of erythrocytes and adjusting the affinity of erythrocyte hemoglobin.Nitric oxide（NO）plays an important role in the physiological and pathological conditions of the body.The smooth muscle relaxation effect of NO plays an important role in the process of hypoxia adaptation and hypoxia injury.Together with O₂,NO can also enter erythrocytes to bind to hemoglobin in pulmonary,and release from hemoglobin in the capillaries of tissues such as brain and muscle.Based on the chaperone relationship between NO and O₂ in erythrocytes,we consider that NO may play an important regulatory role in the oxygen-carrying function of erythrocytes.However,the effect of NO on erythrocyte oxygenation and its role in hypoxia adaptation or hypoxia tolerance has not been reported.In this study,we observed the effects of NO on the oxygen-carrying properties of erythrocytes,and contributions of the properties in NO-induced hypoxia tolerance.Purpose:To study the effect of NO on oxygen-carrying capacity of erythrocyte,hypoxia tolerance in mice and the relationship between them.Method:The peripheral blood of the mice was collected and placed in cuvette of the Oxygen Equilibrium Curve Mapping Instrument,and the blood was sufficiently deoxygenated by introducing nitrogen gas,then the air containing different concentrations of NO（0,100,200 ppm）was introduced to fully oxygenate the blood.The oxygen equilibrium curve（OEC）was plotted and the OEC software was used to calculate P50 and Hill for the curves.The OEC in different pH（7.40,7.15,7.65）and different concentrations of NO（0,100,200 ppm）were also plotted.The P50 is used to calculate the sensitivity index（SI）of the OEC to acid and alkali,SI=（P50HCL-P50NAOH）/P50CON×100%.P50HCL,P50CON,P50NAOH is respectively the P₅₀0 of the OEC in PH7.15、7.40、7.65.Mice were randomly divided into five groups（normoxia group,hypoxia group,hypoxia+5ppm NO group,hypoxia+10ppm NO group,hypoxia+20ppm NO group）.Three kinds of gases（N₂,O₂,NO）were introduced into a chamber,the gases were achieved the desired concentration by adjusting the flow meter valves of each gas.The mice were anesthetized and placed in the chamber,and the tails were left outside the chamber.The survival time of the mice were recorded by stopwatch,and the blood oxygen saturation（SO₂）at the tail of the mice was measured by an oximeter.The mice were randomly divided into four groups:normoxia group,normoxia+NO group,hypoxia（5%O₂）group,and hypoxia+NO（20 ppm）group.The mice were intraperitoneally injected with hypoxic probes.After1 h,the mice were placed in the hypoxia chamber.The mice were sacrificed before the obvious convulsions,the brain tissue was removed for immunohistochemistry stain of hypoxia prober and immunoblotting of hypoxia-inducible factor 1αto reflect the degree of hypoxia in brain tissue.Result:1.NO increased hemoglobin oxygen affinityCompared with the control group（0 ppm NO）group,the oxygenation curve of the 100 ppm NO group and the 200 ppm NO group showed a significant left shift,and the higher the NO concentration,the more obvious the left shift.Compared with the 0ppm NO group,the P₅₀0 of the oxygenation curve of the 100 ppm NO group and the 200 ppm NO group was significantly lower（P<0.05）,but the Hill coefficient was not significantly changed（P>0.05）.2.NO enhances the Boer effectCompared with the control group（0 ppm NO）,the SI of the 100 ppm NO group increased significantly（P<0.05）.The increase of the oxygenation curve SI of the 200 ppm NO group was not statistically significant（P>0.05）.3.NO prolongs the survival time of mice in hypoxia environmentUnder hypoxia,compared with the control group（0 ppm NO）,the survival time of the 5,10,20 ppm NO group was significantly prolonged（P<0.05）,and the higher the concentration of NO induced more obvious of the prolongation.Similar effects of NO were observed in survival rate under hypoxia.4.NO reduces hypoxia-induced decrease of blood SO₂The SO₂ of the mice in the hypoxia group was significantly lower（P<0.05）than normoxia group.In hypoxia,compared with the control group（0 ppm NO）,the decrease of SO₂ in the 5,10,20ppm NO group was significantly mitigated（P<0.05）.The higher concentration NO caused the more obvious mitigation effect.5.NO-induced the improvement of SO₂ was related to NO-induced elongation of survival time under hypoxia.In hypoxic environment（5%O₂）,the NO-induced enhancement of the tolerance to hypoxia was represented by the difference（ΔRT）of the survival time between inhaled and non-inhaled NO mice.The NO-induced improvement of blood oxygen was represented by the increase of SO₂（△SO₂）in inhaled NO mice and non-inhaled NO mice.△RT was significantly positively correlated with△SO₂（P<0.05）,and the slope of the regression line increased with the increase of NO concentration.6.NO alleviated the degree of hypoxia in brain tissue under hypoxiaIn the absence of oxygen,NO can increase the theoretical value of arteriovenousoxygendifference,andweakenthelevelof hypoxia-inducible factor 1αin brain tissue.Compared with the normoxic group,the coloration of hypoxic probes in the brain tissue of the hypoxic group was significantly enhanced.Under hypoxic conditions,the coloration of hypoxic probes in the brain tissue of the inhaled NO group was lighter than that of the control group（non-inhaled NO group）.In conclusion:NO can increase the hemoglobin oxygen affinity of the erythrocyte,increase the sensitivity of the oxygenation curve to acid and alkali,and enhance the oxygen carrying capacity of erythrocytes.In hypoxic environment,inhalation of NO can alleviate the decrease of SO₂ and hypoxia degree in brain tissue,further improve the capability of hypoxia tolerance.