The research on free radicals has been very active in recent years and has generated many theories and opinions. How are free radicals generated in the pathophysiological process of extracorporeal circulation? And how can we actively prevent them? The following is a summary.
A. The concept and nature of free radicals
Free radicals are the general term for atomic groups and molecules that contain unpaired electrons in their outer orbitals. Under normal circumstances, the body produces a small amount of free radicals, which are not harmful to the body because of the “antioxidant defense system”. However, when the body is ischemic and hypoxic, the antioxidant system is inhibited, the damaged cells, especially the mitochondrial electron transport system, increase the “univalent leakage”, the complement-polymorphonuclear leukocyte activation, and the oxygen supply during reperfusion, resulting in an explosive increase of OFR. On the other hand, by reacting with membrane lipid peroxidation, it alters the microenvironment of membrane-bound enzymes, receptors and ions, thus altering the normal function of these proteases, causing Ca2+ overload, promoting cross-linking of membrane proteins and phospholipids, and causing irreversible protease degeneration leading to the destruction of cell membrane structure and function.
II. Generation of oxygen radicals in the in vitro circulation
The main sources of oxygen radicals during CPB are
1. Mitochondrial system: It is currently believed that in the mitochondrial respiratory chain, reduced coenzyme Q is the main site of oxygen radical production and the main source of OFR after myocardial ischemia and reperfusion. The longer the duration of ischemia, the more OFR is produced, which eventually causes depletion of mitochondrial antioxidant products.
2, xanthine oxidase system: severe trauma, shock, local ischemia-reperfusion, etc., cause the accumulation of hypoxanthine and xanthine in tissues, the conversion of xanthine dehydrogenase into xanthine oxidase, the increase of xanthine oxidase level in blood, the activation of neutrophils and their pulmonary aggregation, the increase of myeloperoxidase activity, the degradation of ATP into AMP and adenosine, which can make thus create the conditions for OFR production. When reperfusion provides oxygen molecules, xanthine oxidase promotes the reaction of hypoxanthine or xanthine with oxygen molecules to form O2ˉ, H2O2 and urate. It has been found that high concentrations of xanthine oxidase in circulating blood may be an important mechanism for the development of multi-organ failure.
3. Granulocyte system: granulocytes are another important source of OFR production in CPB. Capillary endothelial cells contain a large number of granulocyte adhesion receptors such as CD18, ELAM, ICAM, etc. Activated granulocytes easily bind to the receptors and adhere and aggregate within the capillary bed, further activating the secretion of elastase and generating large amounts of oxygen radicals via respiratory burst. It has been suggested that reactive oxygen species are the most important initiating factor for tissue damage caused by granulocytes. Inactivation of the anti-protease α-AT [6] by reactive oxygen species disrupts the balance between the protease and anti-protease systems, resulting in the expansion of tissue damage. The popular “microenvironment theory” suggests that a closed microenvironment is created between activated adherent granulocytes and vascular endothelial cells, which prevents the entry of surrounding antioxidants and anti-proteases, while a large number of reactive oxidants and proteases produced by adherent granulocytes are released into the microenvironment and the endothelial damage is expanded [7]. At the same time, the oxygen radicals produced by granulocytes, especially reacting with stimulants in the plasma, form chemokines, which promote granulocyte adhesion and aggregation, forming a vicious circle.
4, During ischemia-reperfusion, due to intracellular Ca2+ overload, calcium-dependent phospholipase is activated, which triggers the metabolism of arachidonic acid (AA).AA produces large amounts of O2ˉ and H2O2 through the action of cyclooxygenase and lipoxygenase, which act on the cell membrane to form lipid peroxides, which in turn can accelerate AA, so AA metabolism produces large amounts of OFR , which in turn further promotes AA metabolism and the imbalance of prostacyclin and thromboxane, resulting in a vicious circle.
Under physiological conditions, the automatic oxidation of catecholamines is very slow, so the effect on the body is not significant, but during CPB, due to the stress response, catecholamines are released in large quantities, and often accompanied by acidosis, etc., the oxidation of catecholamines is accelerated. The details are to be studied in the near future.
Third, the damage of oxygen free radicals to vital organs
1, oxygen free radical damage to the heart
The main manifestations of myocardial ischemia-reperfusion injury are.
(1) myocardial oxygen uptake impairment: ischemic myocardium can not fully utilize oxygen after restoration of normal blood flow, as shown by the difference in oxygen content of coronary arterial and venous blood.
(2) Myocardial hemorrhage and injury: Robert et al. found no or insignificant myocardial hemorrhage during coronary artery blockade, while there was often significant visual or microscopic hemorrhage after restoration of blood supply, and the intra-myocardial hemorrhage that occurred early in reperfusion was more severe than that in the persistent infarct area.
(3) Acute swelling of myocardial cells and no reperfusion phenomenon: experiments showed that after blocking coronary blood flow for 15 min, there was no edema in the ischemic myocardium and only mild changes in ultrastructure, and there was still no obvious edema in the endocardium after 40 min of ischemia, while after 2 min of reperfusion, myocardial cells swelled sharply, accompanied by an increase in intracellular sodium and calcium ions and a decrease in potassium and magnesium ions, and severe intracellular edema was seen in the ultrastructure, and mitochondria swelling, microvascular stenosis due to swelling of cardiomyocytes and vascular endothelial cells, further compression of microvessels by ischemic myocardial contracture, microthrombus formation, etc., resulting in partial failure to restore blood flow during myocardial reperfusion.
(4) Heart rate arrhythmias and cardiac decompensation: Heart rate arrhythmias are mainly manifested as ventricular heart rate arrhythmias, including ventricular tachycardia and ventricular fibrillation, which are associated with increased plasma MDA levels and decreased SOD activity. Due to the multifaceted damage to myocardial tissue from direct intracardiac surgery, which includes the effect of OFR, postoperative ventricular compliance, myocardial contractility, blood pressure and cardiac output are often decreased, and in severe cases, the circulation cannot be supported.
2.Oxygen free radical damage to the lung
Lung damage caused by oxygen free radicals depends on the type and activity of oxygen free radicals in lung tissue and involves almost all cells and tissues in the lung.
Pulmonary vascular endothelial cells are the main source of oxygen radicals in the lung and are also the main target of oxygen radical action. When endothelial cells from animals have been used in contact with H2O2, the endothelial cell periphery is deformed and crumpled, intracellular ATP concentration decreases, Ca2+ concentration increases, proteinases are excited, and the structure and function of cells and cell membranes are disrupted. In animal experiments, intermittent vesicle and vacuole formation was found in interstitial capillary endothelial cells of lung. Oxygen radicals damage the mesenchyme by oxidizing hyaluronic acid and collagen, altering the stability and mobility of the mesenchyme, and oxidative inactivation of methionine radicals in mesenchymal α1-antiprotease, neutralizing α1-antiprotease and elastase, reducing protease inhibitors, and even activating protease, leading to disruption of the balance of the protease-antiprotease system. Increases protein sensitivity to protein hydrolases, aggravates lung damage, and increases the permeability of the simplex. Oxygen radicals damage alveolar epithelial cells by discontinuous vacuole formation in type I cells, proliferation of type II cells and fibroblasts, interstitial fibrosis, decreased oxygen exchange rate and lung compliance in the lung, decreased surface active substances, and further decrease in lung function.
Oxygen radicals promote the release of the thromboxane TxB2, which has a vasoconstrictive effect and raises blood pressure. Oxygen radicals also attenuate the inactivation of 5-HT by endothelial cells, increase the activation of angiotensin II and reduce the formation of endothelial relaxing factors. Oxygen radicals react with cell membranes to produce arachidonic acid metabolites that stimulate lipid peroxidation and affect the whole body. Other experiments have confirmed that IgG-mediated immune complex damage is oxygen radical-dependent.
3, oxygen free radical damage to the brain
Free radical reaction is an important mechanism of cerebral ischemic injury. It is believed that cerebral ischemia and reperfusion can also generate more oxygen radicals through xanthine oxidase system and mitochondrial monovalent leakage, triggering lipid peroxidation reaction. Large amounts of free radicals can damage lipids and cell membranes, peroxidize polyvalent unsaturated fatty acids, lead to decreased fluidity and increased permeability of neural cell membranes, swelling of mitochondria, and release of lysosomal enzymes; aldehyde and hydroxyl radicals generated by lipid peroxidation cause degeneration and necrosis of neural cells; free radicals can cross-link protein molecules and break peptide chains, causing neural cells to lose function; in addition, free radicals can also act on neural cell In addition, free radicals can also act on the external matrix of nerve cells, producing extensive brain tissue damage.
Prevention of oxygen free radicals in CPB
1.The natural antioxidant system in the body
There is a natural oxidative and antioxidant balance system in the body, and both insufficient and excessive oxygen radical generation can cause diseases, such as when the body ischemia and hypoxia, the antioxidant system is inhibited, the damaged cells, especially the mitochondrial electron transfer system “univalent leakage” increases, the complement-polymorphonuclear leukocyte activation, the oxygen supply during reperfusion, so that the OFR is burst increase. However, the body has important antioxidant substances such as SOD, peroxidase, catalase and GSH-PX, and also contains plasma copper cyanide, vitamin C and reduced glutathione that precisely regulate the balance of oxidative and antioxidant systems [16].
2, Reduction of oxygen radical production in CPB
(1) Calcium antagonists: there is a consensus that OFR and calcium have an interaction in myocardial reperfusion injury. The addition of verapamil to the stopping fluid inhibits the increase in calcium content and reduces myocardial MDA content during myocardial reperfusion. Administration of diltiazem 15 μg?Kgˉ1?minˉ1 for 30 min before aortic blockade in small pigs significantly reduced the extent of myocardial infarction and reduced arrhythmias. The area of myocardial infarction was reduced from 79±20% to 53±26% in the control group by continuous administration of diltiazem to dogs (P=0.025). Clinical application of nifedipine also achieved good results, suggesting that calcium antagonists have a protective effect on ischemic myocardium. However, nifedipine also has an effect on vascular smooth muscle, and overdose may cause vasodilation and blood pressure drop, which should be noted. Since these drugs mainly prevent Ca2+ overload in cardiomyocytes and mitochondria during ischemia, they should be administered before aortic block and myocardial ischemia.
(2) Iron complexing agents: During ischemia-reperfusion, there are two main sources of OH: (1) Haber-Wiss reaction, which is slow and the OH produced is not enough to cause cell destruction; (2) excessive metal-catalyzed Fenton reaction, which is tens of thousands of times faster than the Haber-Wiss reaction, and the presence of very small amounts of iron ions in the body can induce the Fenton reaction. Desferrioxamine has a very high affinity for Fe3+ and can quickly bind with Fe3+ to form desferrioxamine Fe3+ complex and exclude it, which can effectively inhibit the iron-catalyzed OFR reaction and has the following characteristics: ① small molecular weight, easy to enter the cell and reduce OFR generation by replacing iron and copper at specific sites; ② can be directly used as a free radical scavenger; ③ can inhibit lipids that depend on (3) It can inhibit the lipid peroxidation reactions that depend on iron and copper trituration and reduce the resulting cell membrane damage.
(3) Allopurinol: It reduces OFR production and cell membrane damage by inhibiting xanthine oxidase activity.Coghlan et al. reported that the application of allopurinol to patients undergoing direct intracardiac surgery significantly reduced the number of lipid peroxidation and postoperative positive inotropic drug applications and improved CI.Johnson et al. found that 169 patients in the allopurinol group had significantly better cardiac function than the control group and lower hospital mortality than the control group. Hospital mortality was significantly lower than that of the control group, and no toxic side effects were observed.
(4) Deleukocyte blood reperfusion: Since leukocytes are not only one of the main sources of OFR production in CPB, but also the accumulation of leukocytes in the microvasculature of the ischemic area generates mechanical obstruction, resulting in a continuous reduction of local blood flow to the myocardium and even no reperfusion, deleukocyte blood reperfusion may be beneficial for myocardial protection. Animal experiments showed that the perfusion vascular resistance, CRK, CPK-MB, SOD activity increased, MDA content decreased, and ultrastructural alterations were significantly lighter in the de-leukocyte blood group than in the control group.
(5) Reduced CPB oxygen flow: Experiment found that after high oxygen flow perfusion, plasma MDA and CPK were significantly elevated, mitochondria were swollen, vacuole-like changes, cristae were broken, cardiomyocytes were severely swollen, and abnormal contraction bands were seen; although MDA and CPK were also elevated in the physiological oxygen group, they were significantly lower than those in the high oxygen group, and vacuole-like changes and cristae breaks in mitochondria were extremely rare. The authors concluded that although the ischemic myocardium required oxygen during recovery, the ability of myocardium to utilize oxygen was reduced in the early stage, mainly because the normal utilization of oxygen was impaired by local hypothermia, enzyme conversion and inhibition in the heart, so that the normal tetravalent reduction of oxygen from OFR could not be carried out effectively, while the monovalent reduction of oxygen from OFR was relatively active. In addition, xanthine dehydrogenase is transformed into xanthine oxidase during hypoxia, and the monovalent reduction of oxygen for high oxygen perfusion becomes more active at this time, resulting in a large amount of OFR production.
3.Scavenging of oxygen radicals in CPB
(1) OFR scavengers: animal experiments found that the continuous input of SOD and CAT before ascending aortic block, 1h and 2h after reperfusion, the left ventricular contractility recovered to 43.3±14% and 74.2±8% of the basal value respectively, while the control group was only 12.8±11% and 31.6±7.8%. Clinical application of SOD and CAT also showed significant improvement in myocardial contractility and cardiac function, and plasma CPK concentration was significantly reduced compared with the control group. However, the effect of SOD on anti-OFR is not complete, and even ineffective in some cases, which may be related to the following factors: (1) the molecular weight of SOD is large, so it is not easy to enter the cell; (2) when ischemia-reperfusion is accompanied by acidosis, the rate of O2ˉ auto-disproportionation to form H2O2 increases, and the relative excess of SOD; (3) the main role of SOD is to make O2ˉ disproportionation to form H2O2, and the latter is formed quickly by Tenton reaction in the presence of metal ions. (3) the main role of SOD is to disproportionate O2ˉ to form H2O2, the latter in the presence of metal ions, through the Tenton reaction quickly formed OH, causing more damage to the body. The above results suggest that the application of SOD, with the application of other drugs may be better.
(2) Low-molecular compounds: Vitamin E is an important antioxidant in the body, which can eliminate O2ˉ, OH, 1O2 and other lipid peroxides. The literature reported [25] that plasma thiobarbituric acid-reactive substances (TBArs) were significantly increased after ischemia-reperfusion and accompanied by a decrease in myocardial vitamin E. The increase in TBArs was negatively correlated with myocardial basal vitamin content and positively correlated with the time of circulatory blockade. The extent of myocardial infarction and arrhythmias were significantly reduced by intraperitoneal injection of vitamin E 100 Mg 30 min before ischemia, indicating the protective effect of vitamins on myocardial ischemia-reperfusion injury. Vitamin C is another important OFR scavenger, which exists not only intracellularly but also extracellularly, and can act extracellularly. 250Mg?Kgˉ1 of vitamin C was injected intraperitoneally before transfer in CPB patients, and plasma MDA and CPK-MB were significantly reduced, but large doses can also induce OFR production, which should be noted. Coenzyme Q is a phospholipase antagonist, which can reduce OFR and has anti-OFR effect. Mannitol is a hydroxyl radical scavenger, which can prevent cell membrane peroxidation. 1,6 diphosphate fructose can inhibit lipid peroxidation, inhibit the respiratory burst of neutrophils, and reduce the damage of O2ˉ and H2O2 on ischemic tissues.
(3) Chinese medicinal preparations: Chuanxunzin can reduce myocardial tissue OFR production during reperfusion, protect the membrane system, increase energy metabolism, improve myocardial tissue ATP, creatine phosphate content and SOD activity, protect myocardial cell mitochondrial calcium pump activity, and prevent intracellular calcium overload in myocardium, which has a better myocardial protective effect. Piperine [26] can significantly reduce OFR production during ischemia-reperfusion, improve cardiac function, reduce the incidence of ventricular fibrillation, maintain Na-K-ATPase activity during ischemia, and reduce intracellular Na and Ca content and CPK release during reperfusion. Salvia, ginsenosides and panax ginseng saponins also have good myocardial protective effects.
(4) Others: Naloxone can reduce the fat content in mitochondria and enhance its anti-lipid peroxidation ability, reduce the damage to mitochondrial structure and enzyme function by reperfusion, increase the stability of cell and mitochondrial structure, slow down the accumulation of intracellular H+ and lactate, and reduce the inward flow of extracellular calcium through the opioid peptide receptor antagonism and direct cell membrane effect of naloxone. Hexamethoxazole increased SOD activity, decreased MDA concentration, and significantly reduced reperfusion arrhythmias.