Effect Of Increasing Oxygen Affinity Of Hb On Blood Oxygen Carrying Characteristics And Anti-hypoxia

BackgroundOxygen is necessary for life,However,when the body outside the respiratory dysfunction,or when the body is in the environment of low O₂concentration or partial pressure,resulting in a decrease of O₂ supply,then will lead to abnormal changes of tissue metabolism,function and structure,what’s more,even irreversible damage to the body when serious,or even directly lead to death.Hemoglobin（Hb）,the highly efficient O₂ transport protein,is a classical allosteric protein.Under the allosteric effectors,it can bind more O₂ in the lungs with relatively high O₂ concentration,and then release O₂ in the tissue cells with relatively low O₂ concentration through the transport of red blood cells,If allosteric agents can be used to improve the affinity of Hb oxygen so as to improve the blood’s ability to obtain O₂ during hypoxia,and the release of O₂ will not be hindered in tissue cells,the oxygenation level of tissue cells can be directly improved.In recent years,Voxelotor（GBT440）,a new hemoglobin allogeneic agent,is a small molecule compound that can improve the affinity of Hb oxygen.GBT440 can form a reversible covalent bond with Valine at the N-terminal of Hb molecule chain.However,GBT440 is mainly used to improve the oxygen affinity of Hb for sickle cell anaemia currently,and there has been no exploration on anti-hypoxia in improving the oxygen affinity of Hb,and no research has been conducted on its oxygen carrying characteristics of blood,especially on the O₂ release ability of Hb.PurposeIn this study,the increase of Hb oxygen affinity with GBT440 was used to explore the influence of the increase of Hb oxygen affinity on blood O₂characteristics and its anti-hypoxia effect,so as to open up new ideas and directions for the prevention and treatment of anoxic diseases.MethodsThis study is divided into three parts:The first part,to explore the effects of increased Hb oxygen affinity on blood O₂ carrying characteristics:First,the dose-effect relationships between the dissociation curves（ODCs）and P₅₀ with GBT440 is detected,and the change of ODCs and the sensitivity index（SI）to acid and base,namely the strength of Bohr effect,were detected by adding acid（HCl）and base（NaOH）respectively.After GBT440 was given to SD rats and treated with oxygen（21%O₂）and hypoxia（10%O₂）for 6 h respectively,blood gas values such as arterial oxygen partial pressure（PaO₂）,arterial oxygen saturation（SaO₂）,arteriovenous oxygen pressure differential（Pa-vO₂）and arteriovenous oxygen saturation differential（Sa-vO₂）were measured by blood oxygen analyzer.The second part,to explore the improvement of Hb oxygen affinity on the degree of hypoxia in tissues:Firstly,hypoxyprobe were injected into mice with normal oxygen and hypoxia,and the amount of binding of hypoxyprobe to hypoxia cells in liver,kidney and brain tissues was measured by immunohistochemistry to reflect the degree of hypoxia.Transmission electron microscopy（TEM）was used to observe the pathological features and severity of hypoxic injury of brain nerve cells and capillary ultrastructure.SD rats were given GBT440 then treatmented with normal oxygen（21%O₂）and hypoxia（10%O₂）.The pH value,PCO₂,HCO₃^-and other changes of blood value were detected,and the incidence of metabolic acidosis was calculated according to the compensation formula of respiratory alkalosis.The third part,to explore the influence of increased Hb oxygen affinity on hypoxia tolerance in mice:The survival rate of the mice receiving different doses of GBT440 was measured under the conditions of hypobaric hypoxia（simulated altitude 10000 m）and normobaric hypoxia（3.5%O₂）respectively.After swimming training in plain environment for a week,the mice given GBT440,hypoxia group was placed in the hypobaric hypoxia chamber with simulated altitude of 5000 m for 6 h,and the normoxia group was placed in the plain environment for 6 h.The weight-bearing swimming experiment was carried out in the device to detect the exhaustion time of each mouse swimming.Mice were trained on a runner wheel in plain environment for three weeks,and given GBT440,The hypoxia group was placed in a normobaric hypoxia chamber for 6 h,while the oxygen group was placed in an atmospheric oxygen environment for 6 h.The wheel exhaustion experiment was carried out in the device to test the wheel exhaustion time of each mouse.Result1.In vitro and vivo experiments,ODCs shifted to the left in a dose-dependent manner and P₅₀ decreased in a dose-dependent manner with the increase of GBT440 concentration（P<0.05）;In vitro,ODCs shifted significantly to the right after the addition of acid,and P₅₀ increased（P<0.05）;ODCs shifted significantly to the left after the addition of alkali,and P₅₀ decreased（P<0.05）,while SI decreased with the increase of drug concentration（P<0.05）.2.In the rats blood oxygen parameters detection experiments,When unused GBT440,compared with normoxia group,PaO₂、SaO₂、Pa-vO₂,Sa-vO₂ were significantly lower（P<0.05）,after using GBT440,these changes are obviously improved,compared with hypoxic control group,hypoxia treatment group PaO₂ increased by 26.51%,SaO₂ increased by 10.78%（absolute value increased by 8.55%）,Pa-vO₂ increased by 96.93%,and Sa-vO₂ was nearly doubled（P<0.05）.Two-factor analysis of variance found that there was a significant negative interaction between hypoxia and medication（P<0.05）.3.Metabolic acidosis was found in all hypoxia groups in the test of acid-base balance index.The incidence of metabolic acidosis in the hypoxia group（20%）was significantly higher than that in the hypoxia group（70%）（P<0.05）.4.Though hypoxyprobe experiment and electron microscope,Without GBT440,compared with normoxia group,The degree of hypoxia of liver,kidney,brain tissue in hypoxia group significantly increased,Severe capillary edema occurred in brain tissues,brain neuron edema,organelle damage and other pathological changes;After the use of GBT440,the degree of hypoxia in liver,kidney and brain tissues is significantly reduced,the brain neuron edema and capillary edema is reduced,and the hypoxia injury is significantly reduced.5.When hypobaric hypoxia and normobaric hypoxia,the survival rate of mice can be improved concentration-dependent with GBT440:In hypobaric hypoxia,the survival rate of 30、70、100 mg/kg group mice was significantly higher than that of 0 mg/kg group mice（P<0.01）,The survival rate of mice in the 100 mg/kg group reached 100%.In normobaric hypoxia,the survival rate of 70、100 mg/kg group mice was significantly higher than that of 0 mg/kg group mice（P<0.05）.6.Compared with the hypoxia control group,the swimming exhaustion time of mice in the hypoxia GBT440 group was significantly prolonged（P<0.01）.Compared with the hypoxia control group,the exhaustion time of the runner in the hypoxia GBT440 group was significantly prolonged（P<0.01）,Two-factor anova found that there was significant negative interaction between hypoxia and medication（P<0.01）.Conclusion1.Allosteric agent GBT440 can significantly improve the Hb oxygen affinity of hypoxic mice.Although it weakens Bohr effect,the Hb combined with GBT440 is still sensitive to acid and alkali,so that the Hb can not only bind more O₂,but also release enough O₂,thus improving the oxygen supply of tissues under hypoxia.2.Increased Hb oxygen affinity significantly improved the degree of hypoxia in liver,kidney and brain tissues during hypoxic hypoxia.3.Increased Hb oxygen affinity significantly improved the survival ability of animals and the ability to work under hypoxia conditions,significantly improved the ability to tolerate hypoxia.