Effects Of Nitric Oxide On The Adaptation And Injury Of Erythrocytes And Its Regulatory Mechanism

Hypoxia is a pathological process which associated with insufficient oxygen supply or oxygen barrier caused by various causes.It is an important cause of death of vital organs,such as heart and brain.Previous studies showed that systemic adaptations to hypoxia permits people successfully adjust to the hypoxic environment at high altitudes.In addition to the roles of cardiopulmonary and hematologic responses that support systemic oxygen delivery in human adaptation to hypoxia,RBCs play an undoubtedly role in systemic adaptations to hypoxia,in line with their vital role in oxygen transport and delivery.Usually,nitric oxide（NO）is an integral part of the human physiological response to hypoxia by increasing local blood flow and oxygen delivery to maintain oxygen homeostasis.Consider the role of NO played in regulating RBC membrane mechanical property and oxygen delivery function,we thought NO also participate in RBC adaptation to hypoxia by modulate RBC deformability and oxygen carrying-releasing function.However,the relationship between hypoxia oxidative stress and RBC’s function and the role of NO in it are not clear at present,and the mechanism is also not clear.Therefore,in this research,RBCs are used as research object,and hypoxia as the research condition,combined with the positive effect of NO on the regulation of RBC functions,we investigated the effect of NO on the adaptation and injury of RBC deformability and carrying-releasing oxygen function in the hypoxic environment/blood bank stored.The main research contents and results are as follows:（1）RBC hypoxia adaptation and injury and its mechanism during hypoxiaThe blood samples were given an hypoxic treatment at the appropriate atmospheric gas concentration（95%N₂,5%CO₂）using a gas system designed by our laboratory.RBCs membrane elongation index（EI）and the parameters of RBC carrying-releasing oxygen（P50 and T50）were determined during hypoxia（0–120 minutes）.Results showed that RBCs have enhanced carrying-releasing oxygen capacity and can maintain well deformation function in the early stage of hypoxia（0-45min）.However,with the prolonged hypoxia time（after 45min）,the deformability and carrying-releasing oxygen capacity decreased,suggested that RBCs functions have hypoxia adaptability and ultimate injury during hypoxia.To analyze the mechanism of RBCs function hypoxia adaptation and injury in different hypxoia time,the results suggested that 1)There are significance increase in RBC lipid peroxidation and free thiols of membrane proteins,and decrease in antioxidants after hypoxia 45min;2)Hemolysis occurred and gradually becomes severe after hypoxia 45min,there are significance increase in metHb levels and membrane bound hemochrome,but there is no obvious time dependence;3)Membrane protein band 3 clustered and phosphotyrosine levels increased after hypoxia45min;4)SHP-2 thiol radical crosslinked with band 3 to high molecular weight substance and lose PTP activity,meanwhile,the express of Syk is also significance increased after hypoxia 45min;5)Hypoxia（0-30min）induced an increasing of NO levels both in plasma and intracellular,after hypoxia 45min,NO level no longer increasing in plasma,on the contrary,decreased in inter-RBC,the results suggested that hypoxia-induced NO in the early stage may related with the hypoxic adaptation of RBC function,but further verification is needed.Next,the relationship between NO and RBC function under hypoxia was discussed in depth.（2）The effect and mechanism of NO on RBC hypoxia adaptationWith the aid of the RBC in vitro hypoxia platform in section（1）,we explore the effect of NO on hypoxia RBC deformability and carrying-releasing oxygen function.The results showed that the NO donor sodium nitroprusside（SNP）concentration has a biphasic regulation effect on RBC deformability under hypoxic conditions.In the experimental concentration range,the hypoxic 60 min RBCs with SNP concentration of10^-6M have the largest deformability;The RBC carrying-releasing oxygen dynamic parameter T50 decreased compared with the positive group,suggested that NO improved the releasing oxygen capacity of hypoxia RBC;Hypoxia-induced membrane lipid peroxidation level is significantly reduced,and membrane protein free thiol and GSH levels significant up-regulation by supplement NO;And also supplement NO significantly inhibited Syk expression and SHP-2 oxidative cross-linking,significantly down-regulated hypoxia-induced tyrosine phosphorylation,and thus significantly down-regulated membrane protein band 3 cross-linking;It is suggested that hypoxia-induced RBC oxidative damage and functional decline are closely related to NO levels in the blood.Inhibition the production of hypoxia-induced NO by supplement NOS inhibitor（L-NAME）can significantly decrease the deformability of RBC with hypoxia 60min,and it works best at a concentration of 10^-4M.At this time,the RBC carrying-releasing oxygen thermodynamic parameter P50 has no significant change,and the T50 is significantly increased,suggesting that the RBC release capacity is decreased.Membrane lipid peroxidation level was significantly up-regulation than that of positive control group,membrane protein free thiol and GSH levels were significantly decreased;tyrosine phosphorylation level was not significantly different,but band 3 cross-linking was significantly enhanced.For hypoxia-induced erythrocyte hemolysis,supplement NO can significantly improve it,inhibiting NOS can significantly enhance it;for hypoxia-induced increase in metHb levels,there is no significant difference in supplementation or inhibition of NO.The tyrosine nitration of RBC membrane protein was detected after hypoxia 60min with SNP or L-NAME.The results showed that the optimal concentrations of SNP and L-NAME had no significant effect on hypoxia-induced tyrosine nitration.The results of this part suggest that NO plays an important role in the maintenance of RBC deformability and releasing oxygen capacity under hypoxic conditions.NO play an important role in RBC function hypoxia adaptation by the regulating of the membrane protein free thiol and GSH reducing activity inhibiting hypoxia-induced membrane lipid peroxidation,and combine the regulation of tyrosine phosphorylation to regulate binding stability of membrane protein band 3 with ankryin-spectrin,and then inhibition of band 3 clustering which involving in regulation of erythrocyte membrane structure and functional stability.（3）Effects of NO on the hypoxia adaptation and damage of RBC during blood bank storageOn the basis of the above study,we further investigated the effect of NO on the hypoxia adaptation and damage of RBC during blood bank storage（0-35days）.Results showed that there was no significant decrease in deformability of RBCs in each group at0-7 days,and continued to decrease,which was significantly lower than that in the normal control group.Compared with the positive control group,supplementation with NO significantly improved the RBC deformability,and inhibited NO to significantly reduce the deformability of RBCs in stored 7-21 days.Supplement NO significantly inhibited the erythrocyte membrane lipid peroxidation,and the membrane protein free thiol oxidation and GSH levels decreased significantly in stored 7-21days.The inhibition of NO significantly increased the degree of membrane lipid peroxidation,and the membrane protein sulfhydryl and GSH decreased significantly in stored 7-21days.Similar to the results in hypoxia situation,supplement NO can significantly reduce the clustering of band 3 and the degree of tyrosine phosphorylation in stored 0-14 days,and inhibit NO significantly enhanced the band 3 cross-linking and tyrosine phosphorylation of stored 0-21 days;There was no significant difference in band 3 cross-linking and tyrosine phosphorylation levels between 28-35 days in blood bank storage;Supplementation or inhibition of NO during RBC preservation had no significant effect on the translocation and cross-linking of SHP-2 to RBC membrane protein band 3;but supplement NO significantly inhibited the expression of Syk in stored RBCs,and the inhibition of NO was significantly up-regulated the expression of Syk.The results suggest that inhibition of NO production during RBC storage can promote RBC oxidative stress damage,promote the expression of Syk to increase tyrosine phosphorylation,and increase band 3 oxidative cross-linking,thereby reducing RBC deformability;Moreover,the study further explore the effect of NO on the biological function of RBCs under the condition of blood preservation and hypoxia,and verify the important role of NO in RBC hypoxia adaptation,which provides a reference for the subsequent study of hypoxia adaptation and injury,and provide a reference strategy for the treatment of hypoxic diseases from the perspective of improving RBC function.In conclusion,this topic finds the hypoxia adaptation and regulation mechanism of RBC deformability and carrying-releasing oxygen capacity.It is found that hypoxic environment/blood bank storage may induce RBC membrane protein,GSH sulfhydryl oxidative and membrane lipid peroxidation through oxidative stress,induce oxidative inactivation of SHP-2 and up-regulate Syk expression,and promote tyrosine phosphorylation,thereby result in membrane protein band 3 oxidative cross-linking,impairing membrane structure stability,affecting RBC carrying-release oxygen function which involved in RBC hypoxia injury process.It also preliminarily confirmed the protective effect of NO/SNO on free thiol-reducing activity,and alleviated the membrane lipid peroxidation induced by hypoxia oxidative stress by protecting GSH and protein thiol,and inhibited th tyrosine phosphorylation by regulating the activity of SHP-2 and Syk in hypoxic RBCs.Furthermore,the stability of band 3-ankyrin-spectrin linkage is maintained,and then inhibit the oxidative cross-linking and dysfunction of band 3,ultimately improve and maintain the deformability and carrying-releasing oxygen capacity of hypoxia RBCs,which involving in regulating the RBC hypoxia adaptation under different oxidative conditions.These studies help to understand the important role of NO in systemic hypoxia adaptation and injury process by the regulation of RBC functions.Meanwhile,it provides a new perspective and thought for the prevention and treatment of clinical hypoxia-related diseases from the perspective of molecular biology.