摘要
目的：肢体缺血/再灌注（I/R）是临床外科中经常发生的病理生理过程，如腹主动脉瘤手术中钳夹血管，大血管栓塞再通或损伤修复，断肢再植等，均可造成以肢体损伤为中心的多脏器损伤，甚至多器官功能障碍 (MOD)，而MOD作为术后严重的并发症，是术后死亡的主要原因之一。肢体缺血/再灌注造成远隔器官损伤的病理机制目前尚未明确，氧自由基（OFR）是其中一个重要的损伤因素，它不仅造成组织细胞的脂质过氧化，还可以引起一系列的炎症反应和细胞损伤。对于肢体缺血/再灌注继发远隔器官损伤的治疗方法，目前仍停留于对症处理，并没有有效的预防和根治措施。异丙酚(Propofol)是一种新型的静脉全麻药，具有起效快，消除快，作用可靠的优点，目前已广泛用于临床麻醉及ICU镇静。近年来许多研究表明异丙酚除麻醉作用外，其特殊的酚羟基结构还可与氧自由基结合而清除氧自由基，发挥较强的抗氧化特性，对组织器官具有明显的保护作用。本实验利用大鼠下肢缺血/再灌注产生大量氧自由基导致远隔器官损伤的模型，观察临床麻醉浓度的异丙酚对大鼠下肢缺血/再灌注后肺、肝、肾组织脂质过氧化产物丙二醛（MDA）含量、抗氧化酶超氧化物歧化酶（SOD）活性、组织诱导型一氧化氮合酶（iNOS）及细胞间黏附分子（ICAM-1）表达的影响，结合组织病理改变，进一步探讨异丙酚对肢体缺血/再灌注后远隔
器官损伤的保护作用及其机制，为异丙酚在多脏器保护方面的临床应用提供实验支持。
方法：实验用wistar大鼠36只，体重300~350g。3%戊巴比妥30mg/kg腹腔内注射麻醉，随机分为3组: (1)假手术组 ( sham组，n=12)，只进行手术操作不做其他处理；(2)单纯肢体缺血/再灌注组(I/R组，n=12)， 双下肢缺血4小时，再灌注6小时；(3)异丙酚+肢体缺血/再灌注组(I/R+P组，n=12) 于开放前10分钟静脉注射异丙酚(得普利麻用5%葡萄糖稀释至0.5%) 5mg·kg-1随后持续输注10mg·kg-1·h-1，I/R组输注等量5%葡萄糖1ml·kg-1 随后2ml·kg-1·h-1维持。实验结束，颈动脉放血处死大鼠，取肺、肝、肾组织测定丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性及肺含水量，观察各组织形态学改变(光镜和电镜)及组织诱导型一氧化氮合酶（iNOS）、细胞间黏附分子（ICAM-1）的表达情况。
结果：1、各组织MDA含量变化：I/R组肾组织MDA含量为0.769±0.224 nmol/mg prot，肝组织MDA的含量为0.699± 0.213 nmol/mg prot，二者均明显高于sham组肾组织（0.616±0.139 nmol/mg prot） 、肝组织（ 0.481±0.134 nmol/mg prot）MDA含量，差异有显著性意义（P<0.05）；I/R组肺组织MDA的含量（1.33±0.473 nmol/mg prot）较sham组（0.847±0.15 nmol/mg prot）明显增高，差异有显著性意义（P<0.01）；与I/R组比较，I/R+P组肾组织（0.599±0.122 nmol/mg prot）、肝组织（ 0.502 ±0.200 nmol/mg prot）MDA含量明显降低（P<0.05），肺组织MDA的含量（0.813±0.273 nmol/mg prot）也明显降低，差异有显著性意义（P<0.01）；I/R+P组与sham组相比三种组织中的MDA含量均无差异性
（P>.05）。
2、各组织SOD活性改变：sham组肺组织SOD活性为11.871±1.821 NU/mg prot ，I/R组为7.415±2.315 NU/mg prot，与sham组相比明显降低（P<0.01），I/R+P组肺组织SOD活性为9.841±2.886 NU/mg prot，较I/R组有所增高（P<0.05），但仍然低于sham组（P<0.05）； I/R组肝组织SOD活性为9.402±2.298 NU/mg prot，低于sham组（12.062±2.020 NU/mg prot）（P<0.05），I/R+P组肝组织SOD活性为 11.663±2.786 NU/mg prot，较I/R组增高（P<0.05），与sham组相比无统计学差异（P>.05）；肾组织SOD活性I/R组为7.236±3.445 NU/mg prot，与sham组（13.261±2.626 NU/mg prot）比较明显降低，差异有显著性意义（P<0.01），I/R+P组肾组织SOD活性（11.762±3.438 NU/mg prot）较I/R组增高（P<0.05），与sham组相比，差异无显著性意义（P>.05）。
3、肺含水量（%）改变：sham组肺含水量为（63.5±8.67）%，I/R组为（75.8 ±5.76）%，I/R+P组为（69.4 ±7.54）%。I/R组与sham组相比肺含水量明显增加(P＜0.01)；而I/R+P组较I/R组有所降低(P＜0.05)；I/R+P组与sham组比较，差异无显著性意义(P>0.05)。
4、组织病理变化：（1）肺组织光镜观察，I/R组肺间隔增厚，间质中有大量中性粒细胞浸润，局部肺泡出现不张、肺泡水肿。I/R+P组肺间质仅轻度增宽，少量中性粒细胞浸润无肺不张或肺出血；电镜观察，I/R组部分肺泡上皮细胞缺损，Ⅰ型上皮细胞肿胀，Ⅱ型上皮细胞微绒毛稀疏、板层小体排空，线粒体结构破坏呈空泡状改变；I/R+P组肺组织超微结构明显改善。（2）肝组织光镜观察：I/R组肝细胞呈
严重水变性。I/R+P组细胞肿胀明显减轻。（3）肾组织光镜观察：I/R组肾小管细胞严重水肿，肾小球及球后毛细血管明显扩张，球后毛细血管内堆积有大量破裂的红细胞； I/R+P组肾结构无明显变化。
5、组织中iNOS、ICAM-1表达：I/R+P组肺、肝、肾组织iNOS、ICAM-1表达阳性的细胞较I/R组明显减少。
结论：异丙酚对大鼠下肢缺血/再灌注造成的远隔器官损伤具有一定的保护作用，其机制可能与清除氧自由基，进?
Objective : Hind limbs ischemia/ reperfusion is a commonly occurring event in clinical practice，such as major vascular surgery with aortic cross-clamping ，peripheral arterial occlusion and reperfusion, limb replantation and so on . Reperfusion of limbs always initiates both local and systematic damage. Sometimes it can alsodevelop into multiple organ dysfunction （MOD）, which is the most serious complications following hind limbs ischemia- reperfusion, and this syndrome presents a high mortality rate in patient. Up until now the mechanism of this phenomenon yet clear, many study has indicated that oxygen free radicals playing an important role in the physiopathological of ischemia-reperfusion injury. It not only initiates lipid peroxidation process but also lead to inflammation responds and tissue injury. Despite advances in medical knowledge and technology, little progress has been made in the treatment of such patients. Propofol is a new an intravenous anesthetic, which has gained worldwide application not only as an anesthetic agent but also as a sedative agent in the intensive care unit because it can be easily titrated and offers
the prospect of rapid recovery. It has been shown to exhibit signigicant antioxidant activity and resembles the endogenous antioxidant α-tocopherol(vitamin E) by reacting with free radicals to form a phenoxyl radical in vitro. So Propofol have obvious protective action on organs. In this study, we used propofol on the model of distant organs injury following hind limbs ischemia/ reperfusion in rats to observe the changes of MDA content, SOD activity, and the expression of inducible nitric oxide syntheses (iNOS), intercellular adhesion molecule type 1(ICAM-1) in lung, liver, kidney tissue and combined with the changes of pathology in lung, liver, kidney tissue to discuss the organ protective effects of propofol and its mechanism. We hope this study will offer the experiment support for the application of propofol on organ protection in clinical practice.
Methods: Thirty-six healthy wistar rats weighing 300~350g were randomly assigned into three groups and anesthetized by 3% pentobarbital 30mg﹒kg-1 i.p. : (1) sham group(n=12), the rats received the operation without other treatment; (2) hind limbs ischemia/ reperfusion group (I/R group, n=12), hind limbs 4-hour ischemia followed by 6-hour reperfusion; 3)propofol treated group (I/R+P group, n=12), the rats received a bolus of 0.5% propofol 5mg﹒kg-1 iv and then continuously infused 10mg﹒kg-1﹒h-1 10 minute before reperfusion. The I/R group were administered with
an equivalent dosage of normal saline (NS) alone instead of propofol, that is 1ml·kg-1 bolus followed by 2ml·kg-1·h-1 continuously. At the end of the experiment, all rats were killed by carotid bloodletting, the lung, liver and kidney tissue was sampled to determined the MDA content, SOD activity, the water content of lung, the expression of iNOS, ICAM-1 and have microscopic examination (light and electron microscopy).
Result
The variety of ?MDA in every tissue or organ as follow:
The MDA content of kidney(0.769±0.224 nmol/mg prot)and that of liver（0.699± 0.213 nmol/mg prot）in group I/R?were significantly larger than those (0.616±0.139 nmol/mg prot, 0.481±0.134 nmol/mg prot by turns) in the group sham(P<0.05).The MDA level of lung in group I/R（1.33±0.473 nmol/mg prot）were notably higher than that in group sham（0.847±0.15 nmol/mg prot）（P<0.01）. Compared with the group I/R，the MDA of kidney（0.599±0.122 nmol/mg prot）and that of liver MDA（ 0.502 ±0.2 nmol/mg prot） decreased obviously in group I/R+P（P<0.05）and ?also did that of lung MDA（0.813±0.273 nmol/mg prot）（P<0.01）. After compared I/R+P group with sham group, we found that their content of MDA in these three tissues had no difference by statistis analysis（P>0.05）.
2 The alteration of SOD activity in every tissue
The SOD activity of lung was 11.871±1.821 NU/mgprot in sham group. It was 7.415±2.315 NU/mg prot in group I/R（remarkably lower than group sha
引文
Morsey H, Aslam M, Standfield N. Patients with critical ischemia of the lower limb are at risk of developing kidney dysfunction. Am J Surg., 2003, 185(4): 360~363
Punch J, Rees R, Cashmer B et al. Acute lung injury following reperfusion after ischcmia in the hindlimb of the rat. J Trauma.1994, 31(6): 760
Cohen SM,Siddiqi FA, Darkchiev B, et al. Attenuation of acute lung injury caused by hind-limb ischemia-reperfusion injury by butyrolactone antiinflammatory afent FL1003. J Trauma, 1997, 43(2): 247
Vega VL, Mardones L, Maldonado M et al. Xanthine oxidase released from reperfused hind limbs mediate kupffer cell activation, meutrophil sequestration, and hepatic oxidative stress in rats subjected to tourniquet shock. Shock, 2000, 14(5): 565
Willoughby RP, Harris KA, Carson MW, et al. Intestinal mucosal permeability to 51C1ethylenediam inetetraacetic acid is increased after bilateral lower extremity ischemia -reperfusion in rat. Surgery. 1996, 120(3): 547
Paterson IS, Klausner JM, Pugatch R, et al. Noncardiogenic pulmonary edema after abdominal aortic aneurysm surgery. Ann Surg, 1989, 209(2): 231~236
Fantini CA, Conte MS. Pulmonary failure following lower torso ischemia: clinical evidence for a remote effect of
reperfusion injury. Am Surg, 1995, 61(4):316~319
Welbourn R, Goldman G, Kobzik L, et al. Role of neutrophil adherence receptors (CD 18) in lung permeability following lower torso ischemia. Circ Res, 1992, 71(1): 82~86
Harkin DW, Barros D'Sa AA, et al. Bactericidal permeability increasing protein attenuates systemic inflammation and acute lung injury in porcine lower limb ischemia-reperfusion injury. Ann Surg, 2001, 234(2): 233~244
Edrees WK, Lau LL, Young IS, et al. The effect of lower limb ischaemia-reperfusion on intestinal permeability and the systemic inflammatory response. Eur J Vasc Endovasc Surg, 2003, 25(4): 330~335
Defraigne JO, Pincemail J. Local and systemic consequences of severe ischemia and reperfusion of the skeletal muscle. Physiopathology and prevention. Acta Chir Belg, 1998, 98(4): 176~186
Barry MC, Kelly CJ, Abdih H, et al Differential effects of lower limb revascularisation on organ injury and the role of the amino acid taurine. Eur J Vasc Endovasc Surg. 1997, 13(2): 193~201
Yassin MM, Harkin DW, Barros D'Sa AA, et al. Lower limb ischemia-reperfusion injury triggers a systemic inflammatory response and multiple organ dysfunction. World J Surg, 2002, 26(1): 115~121
Liu D, Jeppssod B, Hakaosson CH, et al. Multiple-system organ damage resulting from prolonged hepatic inflow
interruption,[J]. Arch Surg, 1996, 131: 442~447
Gaty MG, Guice K S, Olddham KT, et al. Evidence for tumor necrosis factor induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury. Ann Surg, 1990, 212: 694~700
金惠明 主编，《病理生理学》第四版人民卫生出版社，2000，146~150
Koksal C, Bozkurt AK, Cangel U, et al. Attenuation of ischemia/reperfusion injury by N-acetylcysteine in a rat hind limb model. J Surg Res, 2003, 15, 111(2): 236-239
Klausner JM, Anner H, Paterson IS, et al. Lower torso ischemia-induced lung injury is leukocyte dependent. Ann Surg, 1988, 208(6): 761~67
Gadaleta D, Fantini GA, Silane MF, et al. Neutrophil leukotriene generation and pulmonary dysfunction after abdominal aortic aneurysm repair. Surgery, 1994, 116(5): 847~852
Goldman G, Welbourn R, Klausner JM, et al. Oxygen free radicals are required for ischemia-induced leukotriene B4 synthesis and diapedesis. Surgery, 1992, 111(3): 287-293
Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol, 2000, 90(3): 255
Sun JS, Lu FJ, Huang WC, et al. Antioxidant status following acute ischemic limb injury: a rabbit model. Free Radic Res, 1999, 31(1): 9~21
沈施仁 陈金 吴信华 等，肢体缺血再灌注时红细胞内
MDA及血浆中NO、TXB2含量的变化, 中华麻醉学杂志，1999，19（5）: 315~316
康一凡 高建军 吴岳嵩等，肢体缺血再灌注损伤局部与主要器官脂质过氧化的观察,第二军医大学报1996，17（5）467~470
Mathru M, Dries DJ, Barnes L, et al. Tourniquet-induced exsanguination in patients requiring lower limb surgery. An ischemia-reperfusion model of oxidant and antioxidant metabolism. Anesthesiology, 1996, 84(1): 14~22
Ikeda.Oxygen free radicals in genesis of traumatic and peritumoral brain edema.Nurosurgy, 1989, 24(5): 679
史中立 凌亦凌 姚玉霞等, 大鼠肢体缺血-再灌注所致肝损伤及其机制探讨，中国病理生理杂志，2001，17（11）：1141
史中立 凌亦凌 姚玉霞等, 大鼠肢体缺血-再灌注所致肾损伤及其机制探讨，中国病理生理杂志，2001，17（11）：1140
扬秀红 张连元 孙树勋等，一氧化氮在大鼠肢体缺血再灌注后肺损伤的作用，生理学报2002，54（3）：234-238
Punch J, Rees R, Cashmer B et al. Acute lung injury following reperfusion after ischcmia in the hindlimb of the rat. J Trauma, 1994, 31(6): 760
Gute DC, Ishida T, Yarimizu K, et al.Inflammatory responses to ischemia and reperfusion in skeletal muscle. Mol Cell Biochem, 1998 ,179(1-2): 169~187
James R, Glen Jb. Synthesis, biological wvalution, and
preliminary structure-activity consideration of a serier of alkyphenols as intravenous anesthetic agents [J]. J Med Chem, 1950, 23: 1350~1357
Smith LB, White PF, Natharson M. Propofol : An update in its clinical use [J] . Anesthesiology, 1994, 81: 1005~1043
Murphy P G, Myers DS, Davies MJ et al. The antioxidant potential of propofol (2.6-dissopropylphenol). Br J Anaesth, 1992, 68: 613~618
Musacchio E, Rizzoli V, Bianchi A et al. Antiocidant action of propofol on liver microsomes, mitochondria and bain synaptosomes in the rat. Pharmacal Toxical, 1991, 69: 75~77
Murphy PG，Davis MJ，Coiumb MO，et al．Effect of propofol and thiopentone on free radical mediated oxidative stress of the erythrocyts．Br J Anaesth，1996，76：536~543
Kokita N，Hara A．Propofol attenuates hydrogen peroxide-induced mechanical and metabolic derangements in the isolated rat heat．Anesthesiology，1996，84：117~127
Eriksson O，Pollesello P，Saris NE．Inhibition of lipid peroxidation in isolated rat liver mitochondria by the general anesthetic propofol．Biochem Pharmacol，1992，44：391~393
Green TR，Bennett SR，Nelson VM．Specificity and properties of propofol as an antioxdant free radical scavenger．Toxicol Appl Pharmacol，1994，129：163-169
Hans P，Deby G，Deby-Dupont G，et al．Effect of propofol on in vitro lipid peroxidation induced by different free radical generating system：a comparison with vitamin E．J
Neursourg Anesthesiol，1996，8：154~158
Ward PA, Tiu GO, Hatherill JR, et al. Systenic compoement activiation lung injury and producte of lipid peroxidation. J Clim Invest, 1985, 76(2):517
袁勤生.超氧化物歧化酶.国外医学分子生物学分册.1982,4(6):276
Uhal B D. Cell cycle kinetics in the alveolar epithelia [J]. Am J Physiol, 1997, 272(6 Pt 1); L1031~L1045
Matthay M A, Folkesson H G, Verkman A S. Salt and water transport acroos alveolar and distal airway epithelia in the adult lung[J]. Am J Physiol, 1996, 270:L487~L503
Folkesson H G, Nitenberg G, Oliver B, et al. Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin[J]. Am J Physiol, 1998, 275:L478~L 490
张波 刘又宁，NF-κB与肺疾病，国外医学生理、病理科学与临床分册，1999，19（1）4~6
Suzuki M, Asako H, Kubes P, et al. Neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules. Microvasc Res, 1991, 42(2): 125~38
魏文清 王荣杰 丛建波等，自由基与细胞信号传导，国外医学生理、病理科学与临床分册，1999（5）329~332
谷振勇 朱铁年 凌亦凌, 过氧亚硝基阴离子与线粒体损伤，国外医学生理、病理科学与临床分册，1999（3）194~197
Mikawa K, Alamatsu H, Nishina K, et al. Propofol inhibits human neutrophil functions. Anesth Analg, 1998, 87:
695~701
Jensen AG, Dahlgren C, Eintrei C. Propfol decreases random and chemotactic stimulated locomotion of human neutrophils in virro. 1993, 70: 99~100
Werner J, Z＇graggen K, Fernandez-del Castillo C, et al. Specific therapy for local and systemic complications of acute pancreatitis with monoclonal antibodies against ICAM-1. Ann Surg, 1999, 229: 834~840
Vogetseder W, Dierich MP. Intercellular adhesion molecule-1(ICAM-1,CD54)is associated with action with actin-filaments.Immunobiology, 1991, 182(2): 143~151
Silvestro L, Ruikun C, sommer F, et al. Platelet-activating factor induced endothelial cell expression of adhesion molecules and modulation of surface glycocalyx, evaluated by electron spectroscopy chemical analysis. Semin Thromb Hemost, 1994,20(2):435~441
Chirstman JW,Lancaster LH,Blackwell TS.Nuclear factor κB:a pivotal role in the systemia inflammatory response syndrome and new target for therapy.Intensive Care Med, 1998.24 (11): 1131 ~1138
Lu S, Wang X, Wen L, Han Z, et al.Effect of superoxide dismutase on adhesion molecules expression in skeletal muscle ischemia/reperfusion injury in rats.中华医学杂志2002 Jun 25;82(12):840~843