A Study On The Effect Of Enhancing Oxygen Supply Efficiency Of Hemoglobin To Alleviate Acute High Altitude Hypoxia Injury

BackgroundRegions at an altitude of>3000 m above sea level account for about one-sixth of the total land area of China.The current 14th Five-Year Plan period（2021–2025）is characterized by promotion of strategic positioning of plateau areas in national defense security,economic construction,and social development of China.With the influx of people into high-altitude areas,acute mountain sickness has become a public health problem frequently faced by the academic community.Acute high-altitude sickness refers to a series of pathological changes such as headache and dizziness caused by hypoxia,which can be life-threatening in severe cases.High-altitude hypoxia is the main cause of acute high-altitude sickness.High-altitude hypoxia is a type of hypotonic hypoxia caused by the low partial pressure of oxygen at high altitudes.Higher altitudes are associated with more severe hypoxia.At present,oxygen inhalation is the basic measure to alleviate hypoxia at high altitudes,but it does not improve hypoxia adaptation and limits people’s ability to live normal life at high altitudes.Premedication with traditional Chinese medicine and hypoxic preconditioning can temporarily relieve hypoxia by improving blood flow and increasing the number of red blood cells（RBCs）;however,such measures can easily further aggravate the burden on the heart and lungs,cause irreversible damage to heart and lung function,and induce other serious pathological symptoms.Therefore,there is an urgent need to develop an ideal method that can effectively alleviate acute high-altitude hypoxia and reduce the occurrence of side effects such as erythrocytosis.Hemoglobin is a key protein that carries and releases oxygen in the body and plays an important role in maintaining the normal physiological activities of the body.The oxygen supply efficiency of hemoglobin is an index system to evaluate the function of hemoglobin,which is affected by hemoglobin–oxygen affinity,the Bohr effect,and theoretical oxygen-release capacity.Our previous study showed that the Tibetan population,which has strong anti-hypoxia ability,has a stronger oxygen-carrying capacity（high oxygen affinity）and a stronger Bohr effect than the plains Han population.In addition,those individuals within the plains population who have stronger oxygen-carrying capacity（high oxygen affinity）exhibit stronger adaptation ability after entering the plateau.Moreover,birds and fish living at high altitudes and in the deep sea tend to have a stronger Bohr effect as a result of evolutionary adaptation to low-oxygen environments.Therefore,enhancing the oxygen supply efficiency of hemoglobin by increasing oxygen affinity and the Bohr effect may be an effective means to alleviate acute high-altitude hypoxia injury.However,effective strategies by which to regulate the oxygen supply efficiency of hemoglobin are lacking.New exogenous ligands,such as RSR-13（efaproxiral）,myo‐inositol trispyrophosphate,and 5-hydroxymethylfurfural,have been developed to regulate the oxygen supply efficiency of hemoglobin,but they have been limited to clinical and preclinical studies because of their poor regulation effects.Voxelotor（GBT440）can bind to sickle hemoglobin and increase hemoglobin–oxygen affinity.It has been used in the study of hypoxia tolerance at high altitudes and achieved certain results.However,the effect of GBT440 on regulating the oxygen supply efficiency of normal hemoglobin in vivo is not ideal,and its effect in alleviating acute high-altitude hypoxia is limited.At present,the effect of enhancing the oxygen supply efficiency of hemoglobin in patients with acute high-altitude hypoxia injury remains unclear.ObjectivesIn view of the above problems,this study was performed to screen new compounds that enhance the oxygen supply efficiency of hemoglobin,analyze the interaction between hemoglobin and ligands,and clarify the mitigation effect of enhancing the oxygen supply efficiency of hemoglobin on hypoxic injury at high altitudes.The overall goal is to provide support for new strategies with which to effectively alleviate acute hypoxic injury at high altitudes.Contents1.Establish a screening system for compounds to enhance the oxygen supply efficiency of hemoglobin and obtain one or two candidate compounds2.Use molecular docking and other methods to explore the molecular interactions that enhance the oxygen supply efficiency of hemoglobin3.Determine the effect of enhancing the oxygen supply efficiency of hemoglobin on acute high-altitude hypoxia injury by using an animal model of acute high-altitude hypoxiaMethods and Results1.Screening of compounds to enhance the oxygen supply efficiency of hemoglobinFirst,a screening protocol for hemoglobin-binding compounds was established.Based on the pre-experimental analysis of different protein–small-molecule interaction systems,surface plasmon resonance detection with multiple gradients is preferred as a screening scheme for the interaction between hemoglobin A（Hb A,also known as adult hemoglobin）and small-molecule compounds.In total,3695 small-molecule compounds from the FDA-Approved Drug Library,Clinical Compound Library,and Chinese Pharmacopoeia Natural Product Library were analyzed for their interaction with Hb A,and 300 compounds were screened out to bind to Hb A in a gradient.Second,an oxygen dissociation assay evaluation protocol was established in vitro.Using an automatic microplate reader in a controlled gas environment,the deoxygenation process of oxyhemoglobin of adult（Oxy Hb A）was dynamically recorded.The effects of compounds obtained by surface plasmon resonance screening on the deoxygenation process of Oxy Hb A were analyzed,and the compounds with enhanced oxygen-carrying capacity of Hb A were further screened.The results showed that compared with the control group（36.4%±6.24%）,the Oxy Hb A ratio in the final stage for two compounds,resveratrol（RSV）and curcumin（CUR）,increased to 55.1%±4.89%（P<0.01）and 60.1%±5.12%（P<0.01）,respectively,which enhanced the oxygen-carrying capacity of Hb A.Third,the effects of RSV and CUR on enhancing the oxygen supply efficiency of hemoglobin were determined.The in vitro effects of RSV and CUR on the hemoglobin oxygen supply efficiency of Hb A and rat RBCs were measured.These effects included the P₅₀ value for oxygen affinity(a lower P₅₀ value was associated with higher oxygen affinity),acid–base sensitivity index（SI）for the Bohr effect,and theoretical oxygen-release capacity（ΔSO₂）at different altitudes.The results showed that RSV reduced the P₅₀ value of Hb A by 24.95%（P<0.01）,increased the SI value by 118.11%（P<0.01）,increasedΔSO₂ for the plains area by 32.40%（P<0.05）,and increasedΔSO₂’for the plateau area by 59.96%（P<0.01）.RSV decreased the P₅₀ value of RBCs by 43.53%（P<0.01）,increased the SI value by 44.15%（P<0.01）,increasedΔSO₂ for the plains area by 18.61%（P<0.05）,and increasedΔSO₂’for the plateau area by 13.39%（P<0.05）.CUR reduced the P₅₀ value of Hb A by 35.16%（P<0.01）,increased the SI value by 118.0%（P<0.01）,increasedΔSO₂ for the plains area by 44.04%（P<0.01）,and increasedΔSO₂for the plateau aera by 69.58%（p<0.01）.CUR reduced the P₅₀ value of RBCs by 52.81%（P<0.01）,increased the SI value by 53.61%（P<0.01）,increasedΔSO₂ for the plains area by 23.68%（P<0.01）,and increasedΔSO₂’for the plateau area by 17.14%（P<0.01）.Both RSV and CUR enhanced the hemoglobin oxygen supply efficiency of Hb A and RBCs.In summary,in this part of the study,we established a screening system for compounds that enhance the oxygen supply efficiency of hemoglobin and identified two compounds,namely RSV and CUR,that enhance the oxygen supply efficiency of hemoglobin.2.Analysis of molecular interactions that enhance the oxygen supply efficiency of hemoglobinFirst,the site and mode of the interactions of RSV and CUR with Hb A were analyzed.Molecular docking of Hb A with RSV and CUR was performed by computer simulation.The results showed that the phenolic ring on the RSV molecule interacted with valine at position 63 on the heme region of the Hb Aα-chain to form a pair ofπ-hydrogen bonds.The hydrophobic or non-polar amino acids as well as positively charged tyrosine and histidine were mainly distributed around the ring.In the docking mode with the optimal affinity score,the affinity between RSV and Hb A was-5.76 kcal/mol.CUR was found to preferably bind to the heme region of the Hb Aα-chain.CUR contains more electronegative oxygen atoms and forms electrostatic interactions with phenylalanine,leucine,valine,histidine,serine,and asparagine in the heme region of the Hb Aα-chain.The affinity between CUR and Hb A in the docking mode with the optimal affinity score was-7.95 kcal/mol.Next,the effects of RSV and CUR on the structural stability of Hb A were analyzed.The melting temperature midpoint（Tm value）of RSV and CUR with Hb A were respectively detected.The Tm value of Hb A was 57.95℃±0.74℃,and the Tm value increased to 58.64℃±0.75℃when the Hb A:RSV molar concentration ratio was 1:1（vs.Hb A,P<0.05）.The Tm value further increased to 59.87℃±0.46℃when the Hb A:RSV molar concentration ratio was 1:3（vs.Hb A,P<0.01;vs.Hb A:RSV molar concentration ratio of 1:1,P<0.05）.The Tm value of an Hb A:CUR molar concentration ratio of 1:1 increased to 58.53℃±0.18℃（vs.Hb A,P<0.05）,and the Tm value of an Hb A:CUR molar concentration ratio of 1:3 further increased to 59.55℃±0.48℃（vs.Hb A,P<0.01;vs.Hb A:CUR molar concentration ratio of 1:1,P<0.05）.These results indicate that RSV and CUR enhanced the structural stability of Hb A.In summary,in this part of the study,the binding of RSV and CUR to Valα63 and multiple electropositive amino acid sites in the heme region of the Hb Aα-chain were found to enhance the structural stability of hemoglobin.These results suggest that the heme region is a potential regulatory target for enhancing the oxygen supply efficiency of hemoglobin.3.Evaluation of the effect of enhancing the oxygen supply efficiency of hemoglobin on acute high-altitude hypoxia injuryRSV and CUR low-concentration（10 mg/kg）,medium-concentration（50 mg/kg）,and high-concentration（100 mg/kg）groups of mice were established,and the mice were intraperitoneally injected with RSV or CUR for 7 consecutive days.The mice were then placed in a closed environment and administered nitrogen gas at a constant rate.The heart rate was reduced to 0 beats/min for 5 seconds as the asphyxia standard,and the asphyxia tolerance time was recorded:46.67±5.54 s for the control group,44.00±8.00 s for the RSV low-concentration group（vs.control group,P>0.05）,71.50±3.73 s for the CUR low-concentration group（vs.control group,P<0.01;vs.RSV low-concentration group,P<0.01）,65.50±2.59 s for the RSV medium-concentration group（vs.control group,P<0.01）,104.0±11.52 s for the CUR medium-concentration group（vs.control group,P<0.01;vs.RSV medium-concentration group,P<0.01）,100.80±7.47 s for the RSV high-concentration group（vs.control group,P<0.01）,and 142.2±21.03 s for the CUR high-concentration group（vs.control group,P<0.01;vs.RSV high-concentration group,P<0.01）.Both RSV and CUR enhanced the animals’tolerance to acute hypoxic asphyxia,and the CUR medium-and high-concentration groups showed the most significant results.A rat model of acute high-altitude hypoxia was used to evaluate the effect of enhancing the oxygen supply efficiency of hemoglobin on alleviating acute hypoxia injury.Through a large multifactor compound simulated medical experiment chamber,a 6000-m altitude environment was simulated,and the effects of CUR at a medium concentration（100 mg/kg）and high concentration（200 mg/kg）on alleviating hypoxia injury was observed.Detection was performed after 7 days of survival.The high concentration of CUR decreased the P₅₀ value（48.57±1.79 to 38.84±3.02 mm Hg,P<0.01）and increased the SI value（57.23%±3.01%to 74.69%±8.70%,P<0.01）in rats with acute high-altitude hypoxia.It also increasedΔSO₂ at a high altitude（46.36%±2.81%to 66.63%±2.62%,P<0.01）.Moreover,the high concentration of CUR prolonged the exhaustion time（1448±221.4 to 2549±213.9 s,P<0.01）and maintained weight gain（-4.08±2.31 to 1.83±1.59 g,P<0.01）.It increased arterial oxygen saturation（84.07%±4.03%to 92.96%±2.00%,P<0.01）and transcutaneous oxygen partial pressure（15.15±3.06 to 29.93±2.92 mm Hg,P<0.01）.Erythrocytosis was inhibited(9.24±2.06 to7.57±0.41×10¹²/L,P<0.01),as was the increase in hemoglobin（228.9±19.53 to 176.3±8.85 g/L,P<0.01）.The alanine aminotransferase level decreased（73.20±27.34 to41.20±7.80 U/L,P<0.01）,as did the aspartate aminotransferase level（185.2±32.26 to112.5±20.07 U/L,P<0.01）.The plasma glucose level increased（6.48±1.21 to 8.51±0.41 mmol/L,P<0.01）,and the plasma creatinine level decreased（75.00±6.68 to 40.75±18.57μmol/L,P<0.01）.The high concentration of CUR also showed a certain protective effect on pathological damage of the brain,heart,and lungs.CUR at 200 mg/kg was shown to effectively enhance the oxygen supply efficiency and alleviate acute high-altitude hypoxia injury in rats.In conclusion,in this part of the study,RSV and CUR were shown to prolong the tolerance time of asphyxia in mice,and 200 mg/kg of CUR effectively enhanced the oxygen supply efficiency of hemoglobin,prolonged the time to exhaustion in swimming,increased the effective oxygen carrying capacity,alleviated the increase in RBCs and hemoglobin,and alleviated the hypoxic damage of organs in rats with acute high-altitude hypoxia.ConclusionsA compound screening system was established,and two candidate small-molecule compounds（RSV and CUR）that can increase the oxygen supply efficiency of hemoglobin were screened out from 3695 small-molecule compounds.Both RSV and CUR bind to the heme region of hemoglobin and enhance the structural stability of hemoglobin.Modification of the heme region may be a potential way to enhance the oxygen supply efficacy of hemoglobin.RSV and CUR can significantly enhance tolerance of acute hypoxia and asphyxia.CUR can effectively enhance the oxygen supply efficiency of hemoglobin in rats with acute high-altitude hypoxia,significantly improve the time to exhaustion in swimming,increase arterial oxygen saturation and transcutaneous oxygen partial pressure,inhibit the compensatory increase in RBCs,reduce organ damage,and effectively alleviate acute high-altitude hypoxia injury.Focusing on the urgent need to improve treatment of acute high-altitude hypoxia injury,this study examined the use of ligands to enhance the oxygen supply efficiency of hemoglobin.The results showed an obvious effect on alleviating acute high-altitude hypoxia injury,thus providing support for the development of technical strategies to effectively alleviate acute high-altitude hypoxia injury.