动脉粥样硬化家兔主动脉左旋精氨酸/一氧化氮途径的变化

目的　通过观察动脉粥样硬化家兔主动脉一氧化氮 (NO)产生及主动脉左旋精氨酸 (L Arg)转运的变化 ,探讨动脉粥样硬化疾病发生的可能机制。方法　 12只家兔分为高脂组及对照组 ,每组 6只 ,分别喂以高脂饮食及普通饮食 6周 ,取血测定血浆胆固醇 (T Chol)、甘油三酯 (TG)及低密度脂蛋白 (LDL)水平 ,测定血浆亚硝酸盐 (NO-2 )含量 ,测定主动脉孵育液中NO-2 含量及主动脉L Arg转运变化。结果　 (1)高脂组T Chol、TG及LDL水平明显高于对照组 ,分别是 18.6倍、2 .4倍及 48.8倍 (P <0 .0 1) ;(2 )高脂组血浆NO-2 含量高于对照组约 2 0 0 % (P <0 .0 1)。主动脉孵育液NO-2 含量高于对照组 17% (P <0 .0 1) ;(3)高脂组主动脉L Arg转运低亲和力Vmax较对照组增加 9倍(P <0 .0 1) ,Km较对照组增加 1.8倍 (P <0 .0 1) ;(4)高脂组主动脉总的一氧化氮合酶 (tNOS)活性较对照组显著增强 ,比对照组高 73% ,诱导性合酶 (iNOS)比对照组高约 2 0 0 % ,原生型合酶 (cNOS)比对照组低 40 % (P <0 .0 1) ;(5 )血浆精氨酸水平两组间无明显差别 (P >0 .0 5 )。结论　动脉粥样硬化时血管组织L Arg/NO系统功能紊乱 ,内皮源性cNOS活性明显降低 ,而非内皮源性的L Arg转运与iNOS活性显著增强 ;血管L Arg/NOS/NO途径的功能紊乱可能参与动脉     

2型糖尿病红细胞L—精氨酸——一氧化氮合成通路受损

目的 探讨2型糖尿病者红细胞左旋精氨酸－一氧化氮（L－Arg-NO)通路的变化及其临床意义。方法 2型糖尿病患者（19例）与健康人（10例）对照。清晨空腹取静脉血,分离提纯红细胞,用放射同位素标记法测定^3H-L-Arg在红细胞转运的动力学特征;纯化红细胞一氧化氮合成酶（NOS）并测定其含量及活性;测定血浆NO产物－硝酸盐和亚硝酸盐（NOx)的含量。结果 2型糖尿病患者（1）红细胞上L－Arg跨膜总转运能力下降,最大转运速度（Vmax)较对照组下降21％（P＜0．01）,米氏常数（Km)较对照组增加18％（P＜0．01）。经Y^ 载体转运的Vmax较对照组下降28％（P＜0．01）,而Km与对照组差异无统计意义（P＞0．05）。经Y^ L转运的Km较对照组增加10％（P＜0．01）,Vmax无明显变化（P＞0．05）。（2）红细胞NOS含量及活性分别较对照组下降15％（P＜0．01）及34％（P＜0．01）。（3）血浆NOx浓度与对照组比降低了17％（P＜0．05）。结论 2型糖尿病时,红细胞L－Arg-NO通路存在多环节障碍,可能导致NO合成减少,使NO介导的血管舒张受损,增加血小板的活性,促进血小板的聚集和粘附,促进糖尿病血管并发症的发生和发展。     

Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase

Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. Nomega-nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4.     

Cardiac allograft vasculopathy and dysregulation of the NO synthase pathway

Cardiac allograft vasculopathy is the most aggressive form of atherosclerosis in humans and is the leading cause of death after the first year of heart transplantation. Endothelial dysfunction is a major contributing factor to the acceleration of coronary vascular disease in these individuals. A reflection of this endothelial dysfunction is the severe impairment in endothelium-dependent vasodilation that occurs early after transplantation. The etiology of this allograft endothelial alteration is multifactorial and may include preexisting atherosclerosis of the graft vessels, reperfusion injury during transplantation, denervation, disruption of the lymphatic system, and acute and chronic immune injury, as well as traditional risk factors for coronary artery disease (hyperlipidemia, diabetes, hypertension, or hyperhomocysteinemia) and pathogens, such as cytomegalovirus. The alteration in endothelial function affects vasomotor tone of the coronary arteries. Evidence indicates that there may be an impairment of endothelial production and/or activity of NO. Because NO is a potent vasodilator, its deficiency would explain the abnormal vasomotor tone in these individuals. In addition, because NO inhibits key processes in vascular inflammation and atherosclerosis, its absence may contribute to the acceleration of transplant vascular disease. Recent studies from our group and others have shed light on the mechanisms of endothelial dysfunction and its importance in cardiac allograft vasculopathy. In addition, the alteration in endothelial function contributes to vascular inflammation and progression of the disease.     

Donor graft adenoviral iNOS gene transfer ameliorates rat liver transplant preservation injury and improves survival

The exact role of inducible NOS (iNOS) in liver ischemia/reperfusion (I/R) injury is controversial. This study was designed to investigate whether donor liver pretreatment with adenovirus encoding iNOS (AdiNOS) ameliorates I/R injury associated with liver transplantation. Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 24 hours' preservation in cold UW. AdiNOS or control gene vector (AdLacZ) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as control. Recipients were sacrificed 1 to 48 hours posttransplantation. An abundant hepatic iNOS protein expression and marked serum NO elevation was observed in the AdiNOS-treated group, without affecting endothelial nitric oxide synthase (eNOS) expression, before harvesting and after OLT. AdiNOS pretreatment markedly improved liver function assessed by serum aspartate aminotransferase/alanine aminotransferase levels and reduced liver necrosis formation. AdiNOS treatment also was associated with reduced ICAM-1 mRNA expression and neutrophil accumulation in the liver graft after OLT compared with untransfected or AdLacZ-treated group. Furthermore, AdiNOS delivery significantly improved transplant survival, compared with AdLacZ or saline controls. AdiNOS pretreatment did not attenuate I/R-induced apoptotic cell death in the liver graft. Administration of a selective inhibitor for iNOS abrogated the protection afforded by AdiNOS pretreatment. In conclusion, donor pretreatment with AdiNOS led to improved liver graft injury and posttransplantation survival. Downregulation of ICAM-1 mRNA and neutrophil infiltration may be associated with the mechanisms by which AdiNOS pretreatment confer the protection against transplant-associated hepatic I/R injury.     

Elevated nitric oxide levels associated with hepatic cell apoptosis during liver injury

Hepatic injury is a major event in liver surgery such as liver transplantation and it always leads to hepatic cell apoptosis. Nitric oxide (NO) is a key signaling regulation molecule. Many researchers have shown that increased NO level can influence liver cell apoptosis by promoting or inhibiting the relative signaling pathways that are involved in the caspase family, Bax/Bcl‐2, mitochondria, oxidative stress, death receptors, and mitogen‐activated protein kinases. Elucidating the relationships between NO and hepatic cell apoptosis is necessary for ameliorating prognosis of liver surgery. This article reviews the newest research progress in the relationships between higher NO levels and hepatic cell apoptosis in liver injury.     

左旋精氨酸对缺血—再灌注损伤肝脏的保护作用

目的探讨左旋精氨酸（L-arginine,L-Arg）对肝缺血－再灌注损伤（hepaticischemia-reperfusioninjury,HIRI）的防治作用及其机理。方法选择HIRI家兔及肝手术患者,观察一氧化氮水平、丙二醛浓度、血栓素B     

左旋精氨酸对缺血-再灌注损伤肝脏的保护作用

目的：探讨左旋精氨酸（L-arginine,L-Arg）对肝缺血－再灌注损伤（hepatic ischemia-reperfusion injury,HIRI）的防治作用及其机理。方法：选择HIRI家兔及肝手术患者,观察一氧化氮水平、丙二醛浓度、血栓素B2和6－酮基－前列腺素F1α含量、血栓素B2/6－酮基－前列腺素F1α比值、ALT活性、肝细胞形态学的变化及L-Arg对上述指标的影响。结果：HIRI期间,NO、6－酮基－前列腺素F1α显著下降,MDA、血栓素B2、血栓素B2/6-酮基-前列腺素F1α及ALT明显升高,肝细胞形态学发生异常变化;使用L-Arg后,上述指标的异常变化显著减轻,其差异有显著意义（Ｐ＜0.05和Ｐ＜0.01＝。 结论：L－Arg通过提高机体NO水平、降低MDA含量及纠正血栓烷 A2与PGI2的平衡,对HIRI有积极的防治作用。     

肝缺血再灌注损伤与细胞凋亡关系的临床研究

目的 通过检测肝缺血再灌注前后肝组织的细胞凋亡情况,探讨临床手术中肝缺血再灌注与细胞凋亡之间的关系,为更好地预防或减轻临床肝脏手术中造成的缺血再灌注损伤(HIRI)提供理论基础.方法 以细胞凋亡测定法(TUNEL 法)测定肝缺血再灌注前后肝细胞的凋亡情况.结果 肝门阻断前与肝门开放时和关腹前肝细胞的凋亡指数各组间的差异有统计学意义(P＜0.01),肝门阻断前肝细胞的凋亡指数高于肝门开放时和关腹前肝细胞的凋亡指数(P＜0.01);肝门开放时肝细胞的凋亡指数高于关腹前肝细胞的凋亡指数(P＜0.01).结论 研究表明肝脏手术中,在肝细胞短时间(15 min左右)缺血后的再灌注损伤中,肝细胞凋亡和缺血再灌注损伤呈负相关,它并不是术后早期肝细胞损伤的一种主要方式.     

Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.     

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr¹⁷ site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na⁺/Ca²⁺exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca²⁺-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca²⁺ loading and release (thapsigargin/dantrolene), mitochondrial Ca²⁺ uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr¹⁷ of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.     

The first Robert Furchgott lecture: from endothelium-dependent relaxation to the L-arginine:NO pathway

Nitric oxide (NO) is released from vascular endothelial cells and fresh vascular tissue in amounts sufficient to account for the biological actions of endothelium-derived relaxing factor. It is synthesized from the terminal guanidino nitrogen atom(s) of L-arginine, a process that is inhibited by NG-monomethyl-L-arginine (L-NMMA). Studies using L-NMMA have shown that NO is constantly generated by the vessel wall to maintain vasodilator tone. The L-arginine:NO pathway has now been identified in a number of other cells and tissues, in many of which it acts as the transduction mechanism for stimulation of the soluble guanylate cyclase.     

氟中毒家免肝细胞凋亡与肝功能损害

目的：观察中毒对家兔肝功损害及肝细胞凋亡的影响,方法：给家兔饮水投氟3个月,采用流式细胞术检测肝细胞凋亡百分率及细胞周期,全自动徨化分析仪检测血清蛋白含量和有关酶活性改变,结果：氟中毒家兔肝细胞凋亡随投氟剂量的增加呈上升趋势,血清白蛋白,总蛋白含量较对照组明显中毒损害的敏感器官。