Kupffer细胞的激活和移植肝再灌注损伤

现在原发性移植肝无功能已成为继排异反应之后第2大引起再移植的原因。同时还有10%～25%的患者移植后出现移植肝原发性功能紊乱[1]。这些都与移植肝冷保存后Ｋｕｐｆｆｅｒ细胞的激活所引起的再灌注损伤有关,本文就Ｋｕｐｆｆｅｒ细胞激活和移植肝再灌注损伤的关系作一综述。一、概述Ｋｕｐｆｆｅｒ细胞是位于肝血窦腔内的巨噬细胞,它寄居于肝血窦内皮细胞之上或之间,并通过突入内皮细胞下Ｄｉｓｓｅ间隙的胞浆突起与肝细胞直接接触。它的粗面内质网和核周被膜呈过氧化物反应特征并富含溶酶体。Ｋｕｐｆｆｅｒ细胞作为与从胃肠道内吸收的微…     

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.     

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。     

肝移植术中应用S-腺苷-L-蛋氨酸对供肝热缺血损伤的影响

目的探讨术中S-腺苷-L-蛋氨酸（SAMe）加入UW液和血浆冲洗液对热缺血损伤供肝及其恢复的影响。方法建立10min热缺血大鼠肝移植模型，分为A组：UW液灌注＋乳酸钠林格氏液冲洗、B组：UW液灌注＋血浆冲洗、C组：SAMe加入UW液灌注＋血浆冲洗和D组：UW液灌注＋SAMe加入血浆冲洗4组，观察肝组织组织病理学变化和电子显微镜下超微结构变化，并检测血清AST和透明质酸。结果C组和D组术后24h血清AST均低于B组（P〈0．05）。A组术后3h和24h血清HA高于B组（P〈0．05），B组复流后3h及24h血清HA均高于C组和D组（P〈0．05）。组织病理学表现B组复流后3h和24h肝细胞损伤和微循环紊乱较C组和D组明显；超微结构表现，A组复流后3h线粒体肿胀，肝窦内皮细胞肿胀，细胞核不规则，可见内皮细胞凋亡，大部分区域肝窦状隙明显狭窄，内皮层结构模糊，红细胞淤积，受压变形，白细胞附壁，可见内皮层完整性破坏；复流后24h，可见线粒体嵴断裂，核融解。B组内皮细胞损伤较A组轻，C组和D组超微结构表现微循环紊乱和肝细胞损伤表现较B组轻。结论供肝切取术中UW液中加入SAMe灌注保存，血浆冲洗液中加入SAMe可改善热缺血供肝微循环，减轻缺血再灌注损伤，并减轻肝细胞热缺血损伤，有利于10min热缺血供肝功能的恢复。     

大鼠肝血窦内皮细胞缺血再灌注损伤机理及防护的研究

目的　研究肝血窦内皮细胞（ＳＥＣ） 缺血再灌注损伤机理及防护方法。方法　采用大鼠肝部分热缺血再灌注模型,并用丹参和异搏定作为实验组,观察缺血４５ ｍｉｎ 及再灌注３０ ｍｉｎ 后ＳＥＣ 超微结构的变化,及其与氧自由基（ＯＦＲ） 和钙离子拮抗剂的关系,并测定血清谷丙转氨酶（ＡＬＴ） ,谷草转氨酶（ＡＳＴ） 值。结果　缺血再灌注后ＳＥＣ 有损伤,但与ＯＦＲ和细胞内钙超载无关;丹参可防护其损伤,而异搏定不能,但二者均可使肝功能损害减轻。结论　ＯＦＲ 和细胞内钙超载不是ＳＥＣ 缺血再灌注损伤的原因;丹参可防护ＳＥＣ 及肝细胞的缺血再灌注损伤;异搏定不能防护ＳＥＣ 缺血再灌注损伤,但可防护肝细胞损伤,保护肝功能。     

内皮素1单抗对肝移植缺血再灌注损伤中肝细胞凋亡的影响

目的探讨细胞凋亡在移植肝缺血再灌注损伤中的作用及内皮素1(ET-1)单克隆抗体对细胞凋亡的影响。方法应用ET-1单抗灌注移植肝，检测血浆与肝组织中ET-1，检测肝功能，测定移植肝组织的MDA及细胞凋亡：结果移植肝再灌注后血中ET-1，谷丙转氨酶(ALT)和肝组织中ET-1与MDA均明显升高，肝细胞凋亡明显增加；应用ET-1单抗后，血中ET-1，ALT和肝组织中ET-1及MDA均降低，肝细胞凋亡亦明显减少。结论ET-1单抗可通过减轻移植肝脂质过氧化反应的作用，从而减少肝细胞凋亡，起减轻到肝细胞损伤，从而保护移植肝。     

大鼠肝内胆管上皮缺血再灌注损伤及丹参的保护作用

在多种肝胆手术特别是肝移植中不可避免地会出现肝脏的缺血再灌注损伤问题，国内外同行对肝脏在冷缺血或热缺血及再灌注后肝细胞、肝窦内皮细胞和Kupffer细胞等病理生理变化和损伤防护方面均做了大量研究工作，但对胆道系统的缺血再灌注损伤却极少有报道。然而，肝内胆管的缺血再灌注损伤及其导致的术后并发症，如胆管炎、肝内胆汁淤积、胆管狭窄、胆管消失综合征等，严重影响着术后肝脏的功能和移植物的存活。     

大鼠无心跳供体热缺血损伤后供肝组织学与超微结构的动态变化

目的动态观察移植肝热缺血损伤后组织学与超微结构的变化特点，预测无心跳供体（NHBD）中供肝耐受热缺血的安全时限。方法实验动物按供肝热缺血时间分别为0、10、15、20、30、45、60min，随机分为7组。按各组条件分别作原位肝移植，动态观察单纯热缺血期和肝移植术后6h、24h、48h各组移植肝组织学、组织化学和超微结构变化规律。结果NHBD的供肝不仅发生热缺血阶段缺血缺氧造成的损伤，在移植肝复流后，也存在再灌注损伤，且再灌注期肝组织损伤的程度与热缺血时间长短有密切关系。热缺血30min以前，移植肝仅出现组织细胞的变性，未见明显坏死。45min组，肝组织可见小灶状的坏死，在小叶中央区首先发生。60min组，肝细胞坏死的范围扩大，呈片状或弥漫性分布；肝窦内皮细胞明显肿胀呈泡状或气球状，肝血窦阻塞，呈微循环障碍。结论大鼠供肝经受30min以内热缺血损伤，肝组织损伤处在可复性阶段。热缺血60min以后，呈现不可逆性的形态学改变。     

常温肝缺血再灌注肝血窦内皮细胞损伤

目的 探讨肝脏常温 因再灌注稆肝血窦内皮细胞的结构变化及其在再灌注损伤中的作用。方法 将４４只大鼠在常温下分别阻断人肝血流２０、４０、６０和９０ｍｉｎ,然后再开放血流２ｈ,制备成缺血再灌注模型,对肝血窦内皮细胞进行扫描和透射电镜观察,正常对照组大鼠５只。结果 肝血流阻断２０、４０ｍｉｎ时内皮细胞受损;血流开放后２ｈ内皮变化可恢复。肝血流肝断６０、９０ｍｉｎ后内皮细胞出现损伤,使部分内皮缺损,上直接     

常温肝缺血再灌注损伤的实验研究

目的：探讨肝缺血再灌注损伤的作用机制。方法：采用大鼠部分肝缺血再灌注模型，将健康雄性SD大鼠24只随机分为三组：A组（手术对照），B组（肝缺血90min），C组（肝缺血90min再灌注120min）。观察每一动物肝组织病理切片；分别检测血浆谷草转氨酶（AST）、谷丙转氨酶（ALT）、乳酸脱氢酶（LDH）、肿瘤坏死因子（TNF－α）、白介素1β（IL-1β）浓度；测定肝组织中髓过氧化物酶（MPO）含量。结果：肝缺血再灌注后，光镜下大鼠肝组织有明显的肝血窦和中央静脉瘀血，内皮细胞及肝细胞普遍水肿变性；C组肝细胞坏死较B组明显；血浆中肝功能酶学指标显著升高，（B、C组与A组比及C组比B组P均＜0．01）；肝组织中MPO活性升高，以再灌注120min组为著（C组比A组P＜0．01）；与血浆中TNF－α、IL-1β的变化趋势相同（TNF－α：C组比A组P＜0．05，IL－1β：B、C组比A组P均＜0．01）。结论：肝脏微循环障碍是肝缺血再灌注损伤的病理基础；TNF－α、IL-1β介导中性粒细胞参与的肝缺血再灌注损伤过程。     

Ischemia and reperfusion injury in liver transplantation

Ischemia/reperfusion (I/R) injury is a multifactorial process detrimental to liver graft function. An understanding of the mechanisms involved in I/R injury is essential for the design of therapeutic strategies to improve the outcome of liver transplantation. The generation of reactive oxygen species subsequent to reoxygenation inflicts tissue damage and initiates a cellular cascade leading to inflammation, cell death, and ultimate organ failure. The accruing evidence suggests that Kupffer cells and T cells mediate the activation of neutrophil inflammatory responses. Activated neutrophils infiltrate the injured liver in parallel with increased expression of adhesion molecules on endothelial cells. The heme oxygenase (HO) system is among the most critical of the cytoprotective mechanisms activated during the cellular stress, exerting anti-oxidant and anti-inflammatory functions, modulating the cell cycle, and maintaining the microcirculation. The activation of toll-like receptors (TLR) on Kupffer cells may provide the triggering signal for pro-inflammatory responses in the I/R injury sequence. Indeed, dissecting TLR downstream signaling pathways plays a fundamental role in exploring novel therapeutic strategies based on the concept that hepatic I/R injury represents a case for host "innate" immunity.     

Kupffer Cell Function in Ischemic and Nonischemic Livers After Hepatic Partial Ischemia/Reperfusion

Hepatic partial ischemic/reperfusion (I/R) injury, in which ischemic and nonischemic areas of the liver are likely to respond to each other after reperfusion, often occurs following hepatobiliary surgical procedures. Kupffer cells (KCs) are considered to play a major role in hepatic I/R injury. To study the activation of KCs in ischemic and nonischemic liver tissues following hepatic I/R, we investigated the superoxide generation and proinflammatory cytokine production of KCs in both liver parts in a rat model of partial hepatic I/R injury. KC superoxide generation in the ischemic and nonischemic lobes was upregulated 6 and 24 h after reperfusion, respectively, and then accelerated. The production of interleukin-1β (IL-1β) by KCs in the ischemic lobes increased during the early and late phases, 6 h and 48–72 h after reperfusion, respectively. A late increase in IL-1β production was also observed in the nonischemic lobes. Production of tumor necrosis factor-α (TNF-α) increased 6–24 h after reperfusion in both lobes. Upregulation of IL-1β mRNA in the ischemic lobes preceded the upregulation of TNF-α mRNA in both lobes. The hepatic partial I/R process results in activation of KCs in ischemic and nonischemic areas of the liver. The KCs are activated during the early phase after reperfusion in the ischemic areas, followed by activation in both the ischemic and nonischemic areas. This could be a cause of liver dysfunction after partial hepatic I/R during surgery. Received: September 9, 1999 / Accepted: September 26, 2000     

枯否细胞和肝脏缺血再灌注损伤

目的 了解枯否细胞在肝脏缺血再灌注损伤中的作用。方法 采用文献回顾的方法对枯否细胞在肝脏缺血再灌注损伤中的作用加以综述。结果 活化后的枯否细胞可产生和释放多种介质直接或间接地影响肝脏微循环。结论 枯否细胞在肝缺血再灌注损伤中发挥了重要作用。     

Inactivation of the small GTPase Rac1 protects the liver from ischemia/reperfusion injury in the rat

BACKGROUND: In ischemia/reperfusion (I/R) injury, a massive generation of reactive oxygen species (ROS) after reperfusion is a critical factor. Rac, a member of the Rho GTPase superfamily, plays important roles in the production of ROS and activation of nuclear factor-kappaB (NF-kappaB) in vitro. However, the exact role of Rac in the ROS production and NF-kappaB activation in vivo after I/R is still obscure. METHODS: We blocked Rac1 activity in the rat liver using adenovirus encoding a dominant negative rac1 mutant (Ad5N17Rac1) and examined whether inactivation of Rac1 could prevent ROS generation in the hepatic I/R injury. Seventy-two hours after the adenoviral infection, hepatic I/R was induced by Pringle's maneuver for 20 minutes, followed by reperfusion in the rats. RESULTS: Ad5N17Rac1 infection significantly attenuated ROS production after reperfusion and suppressed the hepatic injury. Furthermore, N17Rac1 suppressed NF-kappaB activation and messenger RNA expression of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthetase (iNOS). Ad5LacZ, a control adenovirus, had no effect on the induced hepatic I/R injury, nor did it affect NF-kappaB activation. Immunohistochemical analysis of NF-kappaB (p65) revealed that translocation of p65 to the nucleus after reperfusion was blocked in many of non-parenchymal cells (NPCs) and in hepatocytes in the Ad5N17Rac1-infected liver. CONCLUSION: We conclude that Rac1 is required in ROS generation and NF-kappaB activation after hepatic I/R in vivo, and that inactivation of NF-kappaB in NPCs and suppression of ROS generation in NPCs and hepatocytes possibly account for the protective effect of N17Rac1 in this study.     

5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperfusion

BACKGROUND: Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo. METHODS: Primary cultures of rat renal proximal tubular cells, subjected to oxidative stress caused by hydrogen peroxide (H2O2), were incubated with increasing concentrations of 5-AIQ (0.01 to 1 mmol/L) after which PARP activation, cellular injury, and cell death were measured. In in vivo experiments, anesthetized male Wistar rats were subjected to renal bilateral ischemia (45 minutes) followed by reperfusion (6 hours) in the absence or presence of 5-AIQ (0.3 mg/kg) after which renal dysfunction, injury and PARP activation were assessed. RESULTS: Incubation of proximal tubular cells with H2O2 caused a substantial increase in PARP activity, cellular injury, and cell death, which were all significantly reduced in a concentration-dependent by 5-AIQ [inhibitory concentration 50 (IC50) approximately 0.03 mmol/L]. In vivo, renal I/R resulted in renal dysfunction, injury, and PARP activation, primarily in the proximal tubules of the kidney. Administration of 5-AIQ significantly reduced the biochemical and histologic signs of renal dysfunction and injury and markedly reduced PARP activation caused by I/R. CONCLUSION: This study demonstrates that 5-AIQ is a potent, water soluble inhibitor of PARP activity, which can significantly reduce (1) cellular injury and death caused to primary cultures of rat proximal tubular cells by oxidative stress in vitro, and (2) renal injury and dysfunction caused by I/R of the kidney of the rat in vivo.     

Hypoxia‐reoxygenation differentially stimulates stress‐activated protein kinases in primary‐cultured rat hepatocytes

Abstract Organ injury after ischemia and reperfusion (I/R) remains one of the most important limiting factors in liver surgery and transplantation. Oxygen‐free radical (OFR) generation is considered a major cause of this damage. JNK₁/SAPK₁, a member of MAPK family, regulates cell adaptation to stressful conditions. The aim of this study was to determine if hypoxia‐reoxygenation (H/R) can activate JNK₁/SAPK₁ and if OFR are involved in this activation. Primary cultured rat hepatocytes isolated from other liver cells and blood flow were submitted to warm and cold H/R phases mimicking surgical and transplant conditions. JNK₁/SAPK₁ was activated by both warm and cold H/R. Deferoxamine (1 mM), di‐phenyle‐neiodonium (50 μM) and N‐acetylcysteine (10 mM) significantly inhibited this kinase activation.     

Oxygen radicals and matrix metalloproteinases mediate reperfusion liver injury

Many pathological processes involve the breakdown and remodeling of the extracellular matrix, which is mediated by the family of important enzymes known as matrix metalloproteinases (MMPs). One such process is warm ischemia/reperfusion (I/R) injury, the most important cause of dysfunction of liver allografts. We monitored protein expression of MMP-9 by Western blotting in rat liver after I/R. We also monitored changes in total MMP activity in the serum before and after I/R. Ischemia was induced by clamping the common hepatic artery and portal vein for 40 minutes and reperfusing for 90 minutes. Blood samples collected before ischemia and after reperfusion were analyzed for AST, hydroxyl radical, and tumor necrosis factor (TNFalpha). This protocol resulted in a high level of MMP-9 expression in liver tissue. Total MMP activity in serum was also significantly increased. Levels of AST, hydroxyl radicals, and TNF alpha were concomitantly increased. Ilomastat, an MMP inhibitor, attenuated the I/R-induced liver injury. After administration of the oxygen radical scavenger N-acetylcysteine (NAC), total MMP activity was suppressed, and liver injury was again attenuated. These results indicated that reperfusion liver injury induced an increase in MMP-9 protein expression and in serum MMP activity. The protective effects of an MMP inhibitor and NAC indicate that oxygen radical production is involved in MMP expression and liver injury associated with I/R.     

Renal ischemia-reperfusion injury: new implications of dendritic cell-endothelial cell interactions

In renal ischemia/reperfusion (I/R) injury endothelial cells are a main target. The disturbance of endothelial cell physiology leads to endothelial swelling and narrowing of the blood vessel lumen. We attribute this effect to impairment of endothelial cell nitric oxide synthase (NOS). NO is significantly reduced in the course of hypoxia causing dysfunction of the vascular smooth muscle tone. Subsequently to an I/R injury, the inflammatory response results in endothial activation with enhanced dendritic cell (DC) adhesion and migration. Thus, alloreactive leukocytes are recruited to the inflammatory site. Finally, dendritic cell–endothelial cell interactions may play a crucial role in antigen-specific allograft rejection in I/R renal injury. DCs, which activate naïve alloreactive T cells, play a central role in the establishment of alloantigen-specific immunity. In the course of hypoxia rejection is initiated at the activated layer of foreign endothelial cells (EC), which forms an immunogenic barrier for migrating DCs and T cells. Host DCs that bind to postischemic activated ECs invade the allografted tissues, or remain stationary in the subendothelial layer, or transmigrate into lymphoid vessels and secondary lymphoid organs, where they present alloantigens to naïve host T cells. Organ rejection is mediated by host alloreactive T cells, which are activated by donor DCs (direct activation) or host DCs (indirect activation). We hypothesized that DC–EC binding and migration is the first step in the renal I/R injury that mediates allotransplant rejection. We sought to better understand the downstream events of a renal I/R injury by understanding DC binding and migration, thereby seeking new strategies for more specific immunomodulatory interventions. Herein we developed a new allotransplant-rejection model after renal I/R injury.     

Mice lacking the 110-kD isoform of poly(ADP-ribose) glycohydrolase are protected against renal ischemia/reperfusion injury

The role of poly(ADP-ribose) (PAR) glycohydrolase (PARG) in the pathophysiology of renal ischemia/reperfusion (I/R) injury is not known. Poly(ADP-ribosyl)ation is rapidly stimulated in cells after DNA damage caused by the generation of reactive oxygen and nitrogen species during I/R. Continuous or excessive activation of poly(ADP-ribose) polymerase-1 produces extended chains of ADP-ribose on nuclear proteins and results in a substantial depletion of intracellular NAD(+) and subsequently, ATP, leading to cellular dysfunction and, ultimately, cell death. The key enzyme involved in polymer turnover is PARG, which possesses mainly exoglycosidase activity but can remove olig(ADP-ribose) fragments via endoglycosidic cleavage. Thus, the aim of this study was to investigate whether the absence of PARG(110) reduced the renal dysfunction, injury, and inflammation caused by I/R of the mouse kidney. Here, the renal dysfunction and injury caused by I/R (bilateral renal artery occlusion [30 min] followed by reperfusion [24 h]) in mice lacking PARG(110), the major nuclear isoform of PARG, was investigated. The following markers of renal dysfunction and injury were measured: Plasma urea, creatinine, aspartate aminotransferase, and histology. The following markers of inflammation were also measured: Myeloperoxidase activity, malondialdehyde levels, and plasma nitrite/nitrate. The degree of renal injury and dysfunction caused by I/R was significantly reduced in PARG(110)-deficient mice when compared with their wild-type littermates, and there were no differences in any of the biochemical parameters measured between sham-operated PARG(110)(-/-) mice and sham-operated wild-type littermates. Thus, it is proposed that endogenous PARG(110) plays a pivotal role in the pathophysiology of I/R injury of the kidney.