环氧二十碳三烯酸在心肌缺血/心肌缺血再灌注损伤中的保护性作用机制研究进展

环氧二十碳三烯酸(epoxyeicosatrienoic acid,EETs)是花生四烯酸(arachidonic acid,AA)在细胞色素P450表氧化酶(Cytochrome P450epoxygenases)作用下生成的一类化合物。在包括人在内的诸多哺乳动物多个器官都发挥重要的生物学作用。在心脏和冠脉,EETs通过调节离子通道、调节基因表达、激活信号转导、与受体结合以及作用于细胞特定位置等诸多方式,发挥着舒张血管、减小冠脉循环阻力、促进心肌细胞缺血后功能恢复、抗凋亡等诸多生物学效应。本文主要就EETs在心肌缺血/缺血再灌注损伤中产生的保护性作用机制及研究进展综述如下。

耳蜗缺血／缺血再灌注与细胞凋亡

细胞凋亡在生物体内广泛存在，研究表明，缺血／缺血再灌注损伤与细胞凋亡有密切的联系，本文仅对耳蜗缺血／缺血再灌注中细胞凋亡的发生、影响因素、调控机制及防治进行综述。

The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury

A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cyto-plasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion （I/R） injury. This tightly orchestrated cata-bolic cellular‘housekeeping’ process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protec-tive mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the va-riability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin （mTOR） inhibitors, adenosine monophosphate-activated protein kinase （AMPK） modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic ma-nipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

CHANGES OF ENDOTHELIN－1 GENE EXPRESSION IN RAT BRAINS DURING ISCHEMIA AND ISCHEMIC REPERFUSION

Objective. The experiment was designed to study the association of cerebral ischemia and reperfusion with endothelin- 1 (ET- 1) gene expression of rat brains and time-dependent changes of ET- 1 gene expression during cerebral ischemia.Materials and methods. Thirty- three male SD rats were divided into dot blot hybridization(n = 27) and in silu hybridization groups(n= 6). The focal cerebral ischemia and reperfusion models were made with suture embolism of middle cerebral artery. Dot blot hybridization groups were redivided into control and ischemic subgroups (ischemia for 0. 5 , 1 , 1. 5 , 3 , 6 , 12 , 24 , 48 and 72 h respectively). In situ hybridization groups were redivided into ischemia and reperfusion groups. After 24 h ischemia and 24 h reperfusion,ET1 gene expressions were investigated with in situ hybridization and the resuhs were analyzed with IBAS 2000 Image Analysis System.Results. Dot blot hybridization showed that ET-1 mRNA of cerebral cortex and caudate- putamen was increased at 6 h of ischemia and reached peak at 24 h (3. 9 and 3. 7 fold respectively) ,and at 72 h of ischemia it remained at high levels(3. 5 and 2. 1 fold respectively). In silu hybridization showed that the levels of ET- 1 mRNA of cerebral cortex and caudate-putamen were also markedly increased both in 24 h ischemia and 24 h reperfusion groups (P<0. 01 , P<0. 05 respectively) .Conclusions. ET-1 gene expression in focal ischemic brain tissue were markedly and progressively increased during cerebral ischemia and reperfusion and downregulation of ET- 1 gene expression may be a new approach to the treatment of ischemic cerebrovascular diseases.

Role of ischaemic preconditioning in liver regeneration following major liver resection and transplantation

Liver ischaemic preconditioning （IPC） is known to protect the liver from the detrimental effects of ischaemic-reperfusion injury （IRI）, which contributes significantly to the morbidity and mortality following major liver surgery. Recent studies have focused on the role of IPC in liver regeneration, the precise mechanism of which are not completely understood. This review discusses the current understanding of the mechanism of liver regeneration and the role of IPC in this setting. Relevant articles were reviewed from the published literature using the Medline database. The search was performed using the keywords ＂liver＂, ＂ischaemic reperfusion＇, ＂ischaemic preconditioning＂, ＂regeneration＂, ＂hepatectomy＂ and ＂transplantation＂. The underlying mechanism of liver regeneration is a complex process involving the interaction of cytokines, growth factors and the metabolic demand of the liver. IPC, through various mediators, promotes liver regeneration by up-regulating growth-promoting factors and suppresses growth-inhibiting factors as well as damaging stresses. The increased understanding of the cellular mechanisms involved in IPC will enable the development of alternative treatment modalities aimed at promoting liver regeneration following major liver resection and transplantation.

INFLUENCE OF LOCAL INFILITRATION ANESTHESIA OF SCALP-POINT ON ACUPUNCTURE INDUCED CHANGES OF CEREBRAL BLOOD PERFUSION DETECTED BY SPECT

Objective: To observe the influence of local infiltration anesthesia at the scalp point on acupuncture stimulation induced changes of cerebral blood perfusion in the brain. Methods: Experiments were conducted in 10 healthy volunteer subjects (8 males and 2 females) who were ordered to take a lying position on a specific bed with their heads keeping in a fixed position. Scalp point used was Motor Area (MS 6). The first syringe needle (gauge 5) was inserted into the scalp from the upper 1/5 of MS 6 and the second syringe needle inserted into the scalp from the middle 2/5 of MS 6 and advanced downward, with the two needles connected to a HAN’s Therapeutic Apparatus. Images of the brain were then taken before and after electroacupuncture (EA) stimulation, and after local injection of 1% lidocaine [mixed with epinephrine (1∶200,000, 3 mL)] plus EA by using Siemens ECAM/ICON Single Photo Emission Computed Tomography (SPECT). Intravenous injection of Ethyl cysteinate dimmer 555 MBq was performed before displaying cerebral images. Data of blood functional changing rat (BFCR%) were analyzed quantitatively using a mathematic model. Results: Before EA stimulation, the blood perfusion and function of cerebral cortex, thalamus, basal ganglion and cerebellum on both sides of the brain were basically symmetry. Following EA of MS 6, BFCR% of the contralateral thalamus, parietal lobe and the frontal lobe increased significantly. Following local infiltration anesthesia, BFCR% of the contralatral thalamus declined markedly (P<0.05). The results of quantitative analysis were in agreement with those of visual observation. Conclusion: Local infiltration anesthesia of the scalp point can significantly weaken or block EA stimulation induced changes of BFCR%.

Effect of Intracoronary Infusion of Bone Marrow Mononuclear Cells or Peripheral Endothelial Progenitor Cells on Myocardial Ischemia-reperfusion Injury in Mini-swine

Objective To simulate and assess the clinical effect of intracoronary infusion of bone marrow mononuclear cells or peripheral endothelial progenitor cells on myocardial reperfusion injury in mini-swine model. Methods Twenty-three mini-swine with myocardial reperfusion injury were used as designed in the study protocol. About （3.54±0.90）×10^7 bone marrow mononuclear cells （MNC group, n=9） or （1.16± 1.07）× 10^7 endothelial progenitor cells （EPC group, n=7） was infused into the affected coronary segment of the swine. The other mini-swine were infused with phosphate buffered saline as control （n=7）. Echocardio- graphy and hemodynamic studies were performed before and 4 weeks after cell infusion. Myocardium infarc- tion size was calculated. Stem cell differentiation was analyzed under a transmission electromicroscope. Results Left ventricular ejection fraction dropped by 0% in EPC group, 2% in MNC group, and 10% in the control group 4 weeks after cell infusion, respectively （P〈0.05）. The systolic parameters increased in MNC and EPC groups but decreased in the control group. However, the diastolic parameters demonstrated no significant change in the three groups （P〉0.05）. EPC decreased total infarction size more than MNC did （1.60±0.26 cm2 vs. 3.71±1.38 cm2, P〈0.05）. Undermature endothelial cells and myocytes were found under transmission electromlcroscope. Conclusions Transplantation of either MNC or EPC may be beneficial to cardiac systolic function, but might not has obvious effect on diastolic function. Intracoronary infusion of EPC might be better than MNC in controlling infarction size. Both MNC and EPC may stimulate angiogenesis, inhibit flbrogenesis, and differentiate into myocardial cells.

Intestinal ischemia and reperfusion induced second impairment of the rat heart

Objective: To study the changes of oxygen free radical, expression of apoptotic gene, ultrastructure of myocardial cell and second injury of the heart following the intestinal ischemia and reperfusion. Methods: Making the models of ischemia and reperfusion by clamping superior mesenteric artery, the concentration of NO and SOD in the blood was examined before clamping the artery and reperfusion for 0, 30, 60 min, 1, 3, and 7 d. The expression of Bax, Bal-2, and p53 in myocardial cell was studied by means of immunohistochemical SP method and the microstructure damage was observed under electron microscopy. Results: After clamping the superior mesenteric artery and reperfusion the concentration of NO increased continuously and reached a peak for reperfusion 7 d (P<0.01) but that of SOD decreased from 30 min to 7 d. The expression of Bax, p53 and Bcl-2 also increased obviously especially for reperfusion 30 min and 7 d following ischemia and reperfusion. After reperfusion for 30 min the positive cell rate of Bax, p53 and Bcl-2 was 53.6%, 45.9% and 67.9%, for reperfusion 7 d it was 52.4%, 43.4% and 31.9% respectively, but the positive cell rate of Bax and p53 was higher than that of Bcl-2. The result of electron microscopy observation showed mfofiliments breaked, dissolved and chromatin condensed. Conclusion: Intestinal ischemia and reperfusion of rat can induced the changes of oxygen free radical and the expression of apoptotic gene and second injury of myocardial cells.

Pretreatment effect of adenosine on activation of NF-κB and level of TNF-α during myocardial ischemia and reperfusion in rats

Objective: To study the pretreatment effect of adenosine on NF κB nuclear activity in ischemia/reperfusion myocardium in rats. Methods: Eighteen healthy male S D rats were randomly divided into three groups. Group I was the control group. The other two groups were subjected to 30 minutes of ischemia, followed by 2 hours of reperfusion. In group III, adenosine was given 40 μg·kg -1 ·min -1 30 minutes before coronary artery occlusion. The NF κB in nuclear was extracted and measured with western blot analysis. TNF α levels in myocardium were measured with enzyme linked immunosorbent assay (ELISA) system. All the data were recorded with mean±SEM, differences at the 95% confidence level were considered significant. Results: NF κB activity in the nucleus significantly increased after ischemia/reperfusion and TNF α levels changed. Adenosine significantly inhibited NF κB activity in nucleus, and concomitantly decreased the level of TNF α in myocardium. Conclusions: Adenosine modulation of NF κB activation may be the cellular molecular mechanism of decreasing of TNF a. The cardioprotective action of adenosine may be involved in the differential modulation of NF κB activation during myocardial ischemia/reperfusion.

Change and role of heme oxygenase-1 in injured lungs following limb ischemia/reperfusion in rats

Objective: To study the change and role of heme oxygenase-1 (HO-1) in injured lungs following limb ischemia/reperfusion in rats. Methods: A total of 96 healthy male Sprague-Dawley rats, weighing 250-300 g, were used in this study. Hind limb ischemia was made on 40 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, then limb reperfusion for 0, 4, 8, 16 and 24 hours (n=8 in each time point) was performed, respectively. Other 8 rats undergoing full surgical operation including isolation of the infrarenal aorta without occlusion were taken as the sham operation group. Lung tissues were obtained from the 48 animals and Northern blotting and Western blotting were employed to measure the changes of HO-1 mRNA and protein expression, respectively. Immunohistochemistry technique was used to determine the cell types responsible for HO-1 expression after limb ischemia/reperfusion. Then hind limb ischemia was made on other 12 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, among whom, 6 rats were given zinc protoporphyrin (ZnPP), an inhibitor of HO. Then limb reperfusion for 16 hours was performed on all the 12 rats. And other 12 rats underwent full surgical operation including isolation of the infrarenal aorta without occlusion, among whom, 6 rats were then given ZnPP. Then lung tissues were obtained from the 24 animals and lung injury markers, lung histology, polymorphonuclear leukocyte (PMN) count and malondialdehyde (MDA) content were detected, respectively. HO activity was determined through measuring the carboxyhemoglobin (COHb) level in artery blood with a CO-oximeter after limb ischemia/reperfusion. And the animal mortality was observed on the other 24 rats. Results: Northern blotting analysis showed that HO-1 mRNA increased significantly at 4 hours after reperfusion, peaked at 16 hours, and began to decrease at 24 hours. In contrast, no positive signal was observed in the sham and simple ischemia animals. Increased HO-1 mRNA levels were accompanied by similar increases in HO-1 protein. Lung PMNs and MDA content increased significantly at 4, 8, 16 and 24 hours after reperfusion, compared with the sham controls (P< 0.001), while they decreased in rats with reperfusion for 16 hours when compared with rats with reperfusion for 4 hours (P< 0.001). Immunohistochemical studies showed that HO-1 was expressed in a variety of cell types, including the airway epithelia, alveolar macrophages and vascular smooth muscular cells. The blood COHb level and animal mortality increased significantly after limb ischemia/reperfusion compared with the sham controls (P< 0.001). ZnPP administrated to the ischemia/reperfusion animals led to a decrease in the COHb level and an increase in lung PMN number, MDA content and animal mortality (P< 0.001 compared with ischemia/reperfusion group), and the lung injury was aggravated. Conclusions: Limb ischemia/reperfusion up-regulates pulmonary HO-1 expression, which serves as a compensatory protective response to the ischemia/reperfusion-induced lung injury in rats.

Protective effect of hemoglobin-induced heme oxygenase-1 on injured lungs caus ed by limb ischemia-reperfusion in rats

To determine the role of hemoglobin( HB) induced heme oxygenase 1 (HO 1) in injured lungs caused by limb ischemia reperfusion (I/R) in rats. Methods: A rat model of ischemia in the hind limbs was made by clamping the infrarenal aorta with a microvascular clip, and lung injury occurre d after reperfusion. To induce the expression of HO 1 in the lungs, Hb was admi nistrated intraperitoneally at 16 hours before reperfusion. Northern blotting an d Western blotting were used to detect the expression of HO 1 in the lungs, and the carboxyhemoglobin (COHb) level in arterial blood was assayed. The effect of h emoglobin (Hb) on the injured lungs after limb I/R was determined by measuring t he changes of lung histology, polymorphonuclear (PMN) count, malondialdehyde (MD A) content and wet to dry weight ratio (W/D). Zinc protoporphyrin (ZnPP), an i nhibitor of HO, was used to determine whether HO 1 was induced by Hb after lun g injury. Results: Hb led to a significant increase in HO 1 mRNA and pro tein expression in the lungs, accompanied by the increase of COHb level in arter ial blood. Compared with the sham controls, the lung PMN count, MDA content and W/D significantly increased at 4 hours after limb I/R, which reversed by th e pretreatment with Hb at 16 hours before reperfusion. ZnPP blocked this protect ive role of Hb in the injured lungs. Conclusions: Hb can induce the lung HO 1 expression, which pla ys an important role in the defense against I/R induced lung injury in rats.

Amino acids protects against renal ischemia-reperfusion injury and attenuates renal endothelin-1 disorder in rats

Objective:To investigate nephroprotective effec ts of a mixture of 8 L-amino acids on renal ischemia-reperfusion injury and it s effects on renal endothelin-1 (ET-1). Methods:The mixture of 8 L-amino acids includes glycine, alan ine, threonine, serine, valine, leucine, isoleucine and proline. Acute ischemic renal injury was induced by clamping renal pedicle for 45 minutes in rats. Sixt y male Sprague-Dawley rats were randomly divided into 3 groups: a sham-operate d group (Group A, n=8), a control group (Group B, n=26) and an amino acid-treat ed group (Group C, n=26). Amino acids were infused at a rate of 1 ml·100g -1 ·h -1 I hour before ischemia and during 3 hours of the whole reperfusio n. The serum creatinine values, BUN levels, creatinine clearance, urine sodium & potassium excretion, urine lactate dehydrogenase (LDH), the rate of urine flow and histological examination were measured. Renal ET-1 levels were assayed wit h radioimmunological assay (RIA) Results: The creatinine clearance was 471.0 μl/min ± 121 .5 μl/min in Group C and 227.0 μl/min± 27.0 μl/min in Group B 3 ho urs after reperfusion, P< 0.01 ). The urine flow rate w as 63.6 μl/min± 15.2 μl/min in Group C and 24.3 μl/min± 7.7 μ l/min in Group B, P< 0.01 ) 1.5 hours after reperfu sion. The serum creatinine was 85.0 μl/min± 7.7 μmol/L and BUN concent ration 11.4 mmol/L ± 3.9 mmol/L in Group C and 112.7 μmol/L± 19.5 μmol/L and 20.7 mmol/L± 6.6 mmol/L respectively in Group B after 24 hours of reperfusion (P< 0.05 ) . The mean histological score by standards of Paller in kidneys was 108.7 ± 15.7 in Group C, and 16 8.8 ± 14.8 in Group B (P< 0.01 ).The renal ET-1 lev els 15 minute and 3 hours after reperfusion were 7.2 pg/mg± 0.8 pg/mg an d 9.6 pg/ml± 1.0 pg/ml in Group C , and 10.1 pg/ml± 2.8 pg/ml a nd 13.0 pg/ml± 2.7 pg/ml in Group B (P< 0.01 ). Conclusions:The mixture of 8 L-amino acids can provide remar kable protection against renal ischemia-reperfusion injury in rats. This may as sociate with attenuation of renal ET-1 disorder.

Protective effect of ischemic preconditioning on liver

Objective:: To study the protective effect of ischemic preconditioning (IPC) on the hepatic ischemia-reperfusion injury. Methods: The model of rat liver subjected to ischemia/reperfusion (I/R) injury was made. All 24 mice were divided randomly into 3 groups and anesthetized by 2% sodium pentobarbital (30-40 mg/kg). The enzyme activity of AST, ALT, LDH, SOD and the content of LPO were assayed respectively. Specimens were observed under transmission electron microscope. Results: IPC prevented the increase of ALT, AST and LDH in the blood and that of LPO in the tissues (P< 0.05 ), and maintained high level of SOD in the tissues (P< 0.05 ). Conclusions: IPC has protective effect on the liver function.

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels

Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endotheli- um during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly in- creased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation （over 67%）. Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation （over 31%）, which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of plate- let-expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo meseuteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leu- kocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte se- questration in hypoxia-reoxygenated microvessels.