枯否细胞在肝移植手术后缺血再灌注损伤中的作用

目的总结枯否细胞在肝移植术后缺血再灌注损伤中的作用。方法通过复习文献的方法对枯否细胞在缺血再灌注损伤中的作用进行综述。结果枯否细胞是肝内固有的巨噬细胞,肝移植手术后枯否细胞被激活释放一系列炎症介质,包括细胞因子、活性氧中间产物、趋化因子等,启动缺血再灌注损伤(ischemia reperfusioninjury,IRI),使移植肝失功。同时,枯否细胞不仅可以通过释放NO来减轻缺血再灌注损伤,也可以特异性的产生血红素加氧酶-1(heme oxygenase-1,HO-1)来发挥对缺血再灌注损伤的保护作用,并且有研究表明HO-1降解血红素产生的代谢产物一氧化碳(carbon monoxide,CO)也有同样的作用。结论枯否细胞在肝移植术后可以发挥双向性作用,如何减少枯否细胞释放各种有害物质,增加有利物质的表达,是今后预防移植肝后缺血再灌注损伤研究的关键。     

氯化钆抑制枯否细胞对大鼠肝脏缺血再灌注损伤的影响

目的探讨抑制枯否细胞对大鼠肝脏缺血再灌注损伤的影响。方法制作部分肝脏缺血再灌注大鼠模型80只,实验组注射氯化钆,对照组注射生理盐水,检测两组大鼠缺血前、再灌注后5min、1和6h血压、心率的变化,血清转氨酶(AST)、肿瘤坏死因子α(TNFα)和白细胞介素1(IL1)的水平及肝组织超微结构的改变。结果实验组再灌注6h血清TNFα和IL1为(0.475±0.069)μg/L和(0.221±0.056)μg/L,显著低于对照组的(0.831±0.167)μg/L和(0.335±0.127)μg/L(P<0.05),两组血压、心率和AST变化的差异也有统计学意义(P<0.05),实验组大鼠肝脏超微结构的损伤程度轻于对照组。结论抑制枯否细胞活化可减轻肝脏缺血再灌注损伤,枯否细胞在肝脏缺血再灌注损伤中的作用很重要。     

挥发性麻醉药与肝缺血再灌注损伤

肝脏缺血再灌注损伤是一重要临床问题。挥发性麻醉药可能通过抑制枯否氏细胞和中性粒细胞产生上的氧自由基,减轻中性粒细胞和肝细胞内钙超载,抑制中性粒细胞的粘附,提高肝细胞缺血再灌注期间的能量贮备,而对肝脏缺血再灌注损伤有保护作用。     

挥发性麻醉药与肝缺血再灌注损伤

肝脏缺血再灌注损伤是一重要的临床问题。挥发性麻醉药可能通过抑制枯否氏细胞和中性粒细胞产生肝细胞外的氧自由基,减轻中性粒细胞和肝细胞内钙超载,抑制中性粒细胞的粘附,提高肝细胞缺血再灌注期间的能量贮备,而对肝脏缺血再灌注损伤有保护作用。     

肝脏缺血再灌注损伤

肝脏是一易受再灌注损伤的器官，原位缺血再灌注〔１〕、移植肝脏〔２〕、离体肝脏〔３〕及孤立肝细胞（如枯否氏细胞〔４〕和肝实质细胞〔５〕）实验都表明肝脏可产生再灌注损伤。近年来实验表明，肝脏的缺血再灌注损伤与多种机理有关。１氧自由基暴发性形成氧自由基的大...     

枯否细胞与肝脏缺血再灌注损伤

休克、肝脏外伤及肝移植所致肝功能衰竭都与肝脏缺血再灌注损伤有关，其中枯否细胞（ＫＣ）的作用不容忽视。ＫＣ可因补体系的激活和钙超载或噬作用而激活，活化后的ＫＣ除主要释放活性氧外，还可入蛋白酶及各种细胞因子，介导对肝细胞的毒性作用，导致严重的肝脏再灌注损伤。干扰ＫＣ功能的药物可能具有重要的临床应用价值。     

中性粒细胞介导的肝脏缺血再灌注损伤

肝脏的缺血再灌注损伤是肝脏移植术、肝脏部分切除术、失血性和心源性休克等手术和临床疾病所共同经历的一个病理生理过程。近年研究表明 ,肝脏缺血再灌注后的损伤分为两个时相[1] :Ⅰ相 ,主要为枯否细胞 (Kupffercell,KC)激活后 ,连同其所分泌的因子如TNF α、IL 1等引起的损伤[2 ] ;Ⅱ相 ,则是在KC、细胞趋化因子、细胞粘附分子等共同作用下 ,使中性粒细胞 (polymorphnuclearleukocyte,PMN)趋化、粘附聚集、活化所介导的损伤[3 ] 。后者是肝脏缺血再灌注损伤的关键 ,其结果是肝细胞的坏死和凋亡 ,最终引起肝脏功能衰竭 ,甚至全身多…     

核因子κB圈套寡核苷酸减轻大鼠移植肝缺血/再灌注损伤的作用和机制

目的探讨抑制枯否（Kupffer）细胞核因子κB（Nuclearfactor-kappaB，NF-κB）活性对减轻大鼠移植肝缺血／再灌注损伤（IRI）的作用和机制。方法建立大鼠肝移植缺血／再灌注损伤模型。实验分正常对照组、缺血／再灌注组和圈套寡核苷酸组，每组均为8只大鼠。圈套寡核苷酸组于移植术前2d经供者尾静脉注入120μg脂质体包裹的NF-κB圈套寡核苷酸。移植再灌注后2h，取各组受者移植肝分离枯否细胞。凝胶迁移变动分析法（EMSA）检测枯否细胞NF-κB蛋白结合活性，逆转录聚合酶链法（RT—PCR）观察枯否细胞肿瘤坏死因子α（TNF—α）和白细胞介素6（IL-6）mRNA的表达，同时观察肝组织病理及肝功能变化。结果缺血／再灌注组移植肝再灌注后2h，枯否细胞NF-κB活性及TNF-α、IL-6 mRNA表达量较对照组明显升高（P〈0．01）。光镜下肝细胞大量变性、坏死，伴有肝血窦明显淤血，血清丙氨酸转氨酶（ALT）和胆红素总量（TBIL）较对照组明显升高（P〈0．01）。相反，圈套寡核苷酸组枯否细胞NF-κB活性及细胞因子mRNA表达与缺血／再灌注组相比明显下降（P〈0．01），移植肝未见明显病理组织学改变，肝功能明显改善。结论NF-κB圈套寡核苷酸能高效抑制枯否细胞NF-κB活性，并抑制其下游有害细胞因子的产生，从而减轻缺血／再灌注损伤对移植肝的打击和损害。     

兔肝脏冷灌注后去除门静脉淤血对血清内毒素以及肝组织中核因子-κB活性的影响

目前临床肝移植多采用非转流手术,术中阻断门静脉可导致肠道淤血、肠上皮的缺血缺氧,使肠道内环境改变,引起肠道内菌群失调,大量产牛细胞内毒素被吸收入门静脉[1].尽管肝脏枯否细胞可以清除内毒素,但移植肝的枯否细胞功能减退,移植肝成为内毒素损伤的主要靶器官,甚至引起肝功能衰竭[2]."放血疗法"已为多数临床医牛接受,但肝移植中去除门静脉淤血多为经验性,缺乏实验依据.我们建立兔肝脏原位冷灌注模型,观察恢复灌流时去除门静脉淤血对血清内毒素及肝脏再灌注损伤的影响及其作用机制,为临床提供一种安全有效的方法.     

参附注射液对大鼠移植肝脏缺血再灌注损伤的影响

目的探讨参附注射液(Shenfuinjection,SF)对大鼠移植肝脏缺血再灌注损伤的保护作用及机制。方法采用雄性SpragueDawley(SD)大鼠同种异体原位肝移植模型,60只SD大鼠随机平均分成对照组和SF处理组,移植肝脏再灌注3、6、24h取血及肝脏组织检测。结果SF组血清超氧化物歧化酶(SOD)和一氧化氮(NO)水平明显高于对照组(P<0·05),丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、透明质酸(HA)、丙二醛(MDA)、肿瘤坏死因子α(TNF-α)、白介素1(IL-1)、内皮素1(ET-1)水平和肝脏细胞凋亡指数明显低于对照组(P<0·05),肝脏组织形态学改变也轻于对照组。结论参附注射液对移植肝脏缺血再灌注损伤具有防治作用,可能的机制包括抑制枯否细胞激活和氧自由基产生,改善微循环,减少细胞凋亡。     

Prevention of primary nonfunction after canine liver allotransplantation: the effect of gadolinium chloride

PURPOSE: Effective suppression of Kupffer cell function is believed to contribute to the prevention of preservation/reperfusion injury. In this study, effect of gadolinium, a synthetic Kupffer cell suppressant, on the reperfusion injury was examined using a canine partial liver transplantation model. METHODS: About a 70% partial liver segment was harvested and reimplanted in a mongrel recipient dog weighing 20 to 25 kg. Gadolinium chloride (10 mg/kg) was infused via the cephalic vein 24 hours before harvest of the partial liver (gadolinium group, n = 5). Serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and morphologic grading of graft were compared with those of a control group (n = 5). Statistical analysis was done with an independent t-test. RESULTS: Average total ischemic time was 4 hours and 27 minutes. At 1 hour after reperfusion, there were no significant differences in AST, ALP, or LDH levels, or pathologic scores. At 48 hours after reperfusion, AST (P = .03) and LDH (P = .05) levels were significantly lower in the gadolinium group. CONCLUSION: Kupffer cell blockade using gadolinium chloride may be effective to reduce ischemia reperfusion injury, but the effect is not evident at an early stage of reperfusion.     

Inhibition of Kupffer cells reduced CXC chemokine production and liver injury

BACKGROUND: Cytokine production is a critical component of ischemia/reperfusion (IR) injury. In the liver, Kupffer cells produce cytokines and chemokines (i.e., cytokines with chemoattractant properties) that are important mediators in neutrophil recruitment and subsequent hepatocellular injury. Therefore, the role of Kupffer cells in chemokine production in hepatic IR injury was investigated. METHODS: Adult male C57BL/6 mice underwent 90 min of partial hepatic ischemia followed by various reperfusion times (i.e., 0, 1.5, 3, and 6 h). Gadolinium chloride (GC), which inhibits Kupffer cell activity, was administered to mice 48 and 24 h prior to ischemia. The control group received a corresponding volume of normal saline. Plasma levels of the cytokine macrophage inflammatory protein-2 (MIP-2), KC, and tumor necrosis factor (TNF)-alpha and liver mRNA were measured. Liver injury was assessed by plasma level of alanine transaminase (ALT) and histopathology. RESULTS: A reperfusion time-dependent liver injury occurred as indicated by increased levels of plasma ALT and histopathology. The injury was associated with increased plasma TNF-alpha, MIP-2, and KC and their hepatic mRNA expression and neutrophil infiltration into ischemic lobes of the liver. GC treatment significantly reduced the number of Kupffer cells as determined by the immunostained liver tissue sections. The extent of liver injury significantly decreased in GC-treated mice that were associated with decreased levels of plasma ALT, TNF-alpha, MIP-2, and KC and neutrophil infiltration. CONCLUSIONS: This study suggests that Kupffer cells are major contributors to cytokine production in hepatic IR and their modulation may serve as a potential target for therapeutic intervention.     

Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion

BACKGROUND AND AIM: Protease inhibitors attenuate ischemia/reperfusion injury. However, the underlying mechanisms by which protease inhibitors prevent reperfusion injury remain obscure. Neutrophils play an important role in reperfusion injury. We studied the effects of urinary trypsin inhibitor (UTI) on production of the C-X-C chemokine, cytokine-induced neutrophil chemoattractant (CINC), by Kupffer cells during ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in rats by occlusion of the portal vein for 30 min. UTI (50,000 U/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined the inhibitory effects of UTI on in vitro CINC production by peritoneal macrophages in response to neutrophil elastase (NE). RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with UTI blunted this increase in CINC and significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. UTI also decreased neutrophil accumulation in the liver 24 h after reperfusion. In vitro CINC production by Kupffer cells from rats pretreated with UTI 3 h after ischemia/reperfusion was significantly decreased compared to those from untreated animals. UTI reduced NE activity in vitro in a dose-dependent manner, and UTI significantly reduced in vitro CINC production by peritoneal macrophages stimulated with NE. CONCLUSION: UTI reduces the production of CINC by Kupffer cells stimulated with NE, attenuating ischemia/reperfusion injury of the liver.     

HEGPOL: Randomized,placebo controlled,multicenter, double-blind clinical trial to investigate hepatoprotective effects of glycine in the postoperative phase of liver transplantation [ISRCTN69350312]

Background  

Kupffer cell-dependent ischemia / reperfusion (I/R) injury after liver transplantation is still of high clinical relevance, as it is strongly associated with primary dysfunction and primary nonfunction of the graft. Glycine, a non-toxic, non-essential amino acid has been conclusively shown in various experiments to prevent both activation of Kupffer cells and reperfusion injury. Based on both experimental and preliminary clinical data this study protocol was designed to further evaluate the early effect of glycine after liver transplantation.     

Regulation of pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells in endotoxin-enhanced reperfusion injury after total hepatic ischemia

The effects of Kupffer cells on cytokine responses in endotoxin-enhanced reperfusion injury after total hepatic ischemia were investigated in this study. Male rats pretreated with either normal saline solution (NS group) or gadolinium chloride (GdCl(3)) to inhibit Kupffer cell function (GC group) were subjected to 60 min of hepatic ischemia. These animals received either normal saline solution or sublethal doses of endotoxin (1 mg/kg) at reperfusion. In the NS group, endotoxin administration induced an enhanced tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 production 1 h after reperfusion with a subsequent peak of macrophage inflammatory protein-2 (MIP-2) levels, which resulted in a 7-day survival rate of 30%. Despite endotoxin administration, GdCl(3) pretreatment significantly suppressed TNF-alpha and increased interleukin-10 production 1 h after reperfusion, which led to a decline in MIP-2 production and amelioration of functional and structural liver damage with a 7-day survival rate of 80%. Augmented pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells were associated with endotoxin-enhanced reperfusion injury after hepatic ischemia. Kupffer cell blockade has a potential to attenuate the insult via modulation of cytokine responses.     

Kupffer细胞在肝移植缺血再灌注损伤中的双重作用

Kupffer细胞足定居于肝内的巨细胞,在月十移植缺血再灌注损伤中发挥着重要的作用,门静脉恢复血流后刺激Kupffer细胞激活,释放活性氧族、多种炎性介质和细胞因子,对肝脏造成损伤.另一方面又可上调HO-1的表达,保护肝脏缺血再灌注损伤,因此,Kupffer细胞在肝移植缺血再灌注损伤中发挥着双重效应.     

Gadolinium chloride-induced improvement of postischemic hepatic perfusion after warm ischemia is associated with reduced hepatic endothelin secretion

Selective Kupffer cell blockade by gadolinium chloride (GdCl₃) pretreatment of liver donors previously proved to be effective in reducing ischemia/reperfusion injury in rat liver transplants. Physiological mechanisms of this effect have not been specified so far. Vasoactive peptides are involved in liver blood flow regulation. We tested the hypothesis, that hepatic hemodynamic effects of GdCl₃ pretreatment are mediated by intrahepatic endothelin‐1 (ET) secretion in a standardized porcine model of warm liver ischemia and reperfusion. Standardized warm hepatic ischemia (45 min) was induced after laparotomy in intubation narcoses (ITN) by Pringle‐maneuver in pigs (n = 12). Animals were either pretreated with GdCl₃ (20 mg/kg i.v.) or sodium chloride 0.9% (control group) in a randomized manner 24 h before investigation. Relaparotomy was performed at day 7. Before, during ischemia and until 6 h after liver reperfusion, transhepatic blood flow (portal venous + hepatic artery flow) was defined by ultrasonic flow probes and hepatic parenchymous microcirculation evaluated by implanted thermodiffusion electrodes. ET plasma concentrations were analyzed (commercial RIA) at all time points in the hepatic veins after selective canulation. GdCl₃ pretreatment of animals markedly improved hepatic macro‐ and microperfusion before and particularly after warm ischemia. Mean ET plasma concentrations in the hepatic vein were significantly lower before, 6 h and 7 days after ischemia, compared with controls. Kupffer cell destruction by GdCl₃ pretreatment improves hepatic micro‐ and macroperfusion after warm ischemia, thus indicating reduced ischemia/reperfusion injury. Documented reduction of postischemic liver blood flow impairment after GdCl₃ pretreatment could be mediated by a decreased hepatic ET secretion, as hemodynamic effects were associated with significantly reduced ET plasma levels in hepatic veins.     

Gadolinium pretreatment decreases survival and impairs liver regeneration after partial hepatectomy under ischemia/reperfusion in rats

BACKGROUND: Modulation of Kupffer cell functions by treatment with gadolinium chloride protects the liver against reperfusion injury. However, its effect on liver regeneration after hepatectomy under ischemia/reperfusion has not been studied. Using a common clinical ischemia/reperfusion technique, we examined the effect of gadolinium on liver regeneration after hepatectomy in rats. METHODS: After an initial 15-minute ischemia and 15-minute reperfusion, 70% hepatectomy was performed during the second 15-minute ischemia period in gadolinium-pretreated (gadolinium group) and saline solution--pretreated (control group) rats. The 24-hour survival rate, relative liver weight, DNA synthesis rate, and hepatic adenosine triphosphate level were examined immediately after hepatectomy and on postoperative days (PODs) 1, 2, 3, and 7. Serum levels of total bilirubin, glutamic pyruvic transaminase, and endotoxin were also measured. RESULTS: The 24-hour survival rate was significantly lower in the gadolinium group (67%) than in the control group (100%). On POD 1, the relative liver weight and DNA synthesis rate were significantly lower in the gadolinium group than in the control group. On POD 1, serum total bilirubin and endotoxin levels were significantly higher in the gadolinium group than in the control group. Immediately after hepatectomy, the hepatic adenosine triphosphate level was significantly lower in the gadolinium group than in the control group. CONCLUSIONS: Under ischemia/reperfusion, gadolinium pretreatment impairs liver regeneration and energy status after hepatectomy and decreases postoperative survival.     

Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver

BACKGROUND: Ischemic injury of the liver is generally considered to result in necrosis, but it has recently been recognized that mediators of apoptosis are activated during ischemia/reperfusion. This study was designed to characterize the extent and the type of cells within the liver that undergo apoptosis at different periods of ischemia and reperfusion. METHODS: Male Wistar rats were subjected to 30 or 60 min of normothermic ischemia. Liver sections were evaluated at the end of ischemia and at 1, 6, 24, and 72 hr after reperfusion. Apoptosis was determined by DNA fragmentation as evaluated by laddering on gel electrophoresis, in situ staining for apoptotic cells using TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and morphology on electron microscopy. RESULTS: In situ staining of liver biopsy specimens using TUNEL showed significant apoptosis after reperfusion. Sinusoidal endothelial cells (SEC) showed evidence of apoptosis earlier than hepatocytes. For example, at 1 hr of reperfusion after 60 min of ischemia, 22+/-4% of the SEC stained TUNEL positive compared with 2+/-1% of the hepatocytes (P<0.001). With a longer duration of ischemia, a greater number of SEC and hepatocytes became TUNEL positive. An increase in TUNEL-positive cells was also noted with an increasing duration of reperfusion. The presence of apoptotic SEC and hepatocytes was supported by DNA laddering on gel electrophoresis and cell morphology on electron microscopy. Several Kupffer cells were seen containing apoptotic bodies but did not show evidence of apoptosis. Only rare hepatocytes showed features of necrosis after 60 min of ischemia and 6 hr of reperfusion. CONCLUSION: These results suggest that apoptosis of endothelial cells followed by hepatocytes is an important mechanism of cell death after ischemia/reperfusion injury in the liver.