C-Jun N-Terminal Kinase 2 Promotes Graft Injury via the Mitochondrial Permeability Transition After Mouse Liver Transplantation

The c-Jun N-terminal kinase (JNK) pathway enhances graft injury after liver transplantation (LT). We hypothesized that the JNK2 isoform promotes graft injury via the mitochondrial permeability transition (MPT). Livers of C57BL/6J (wild-type, WT) and JNK2 knockout (KO) mice were transplanted into WT recipients after 30 h of cold storage in UW solution. Injury after implantation was assessed by serum ALT, histological necrosis, TUNEL, Caspase 3 activity, 30-day survival, and cytochrome c and 4-hydroxynonenal immunostaining. Multiphoton microscopy after LT monitored mitochondrial membrane potential in vivo . After LT, ALT increased three times more in WT compared to KO (p < 0.05). Necrosis and TUNEL were more than two times greater in WT than KO (p < 0.05). Immunostaining showed a >80% decrease of mitochondrial cytochrome c release in KO compared to WT (p < 0.01). Lipid peroxidation was similarly decreased. Every KO graft but one survived longer than all WT grafts (p < 0.05, Kaplan-Meier). After LT, depolarization of mitochondria occurred in 73% of WT hepatocytes, which decreased to 28% in KO (p < 0.05). In conclusion, donor JNK2 promotes injury after mouse LT via the MPT. MPT inhibition using specific JNK2 inhibitors may be useful in protecting grafts against adverse outcomes from ischemia/reperfusion injury.     

肝细胞凋亡在肝硬化大鼠肝缺血再灌注损伤中的意义

目的 研究肝硬化大鼠肝缺血再灌注（I／R）损伤和硬化肝比正常肝更容易损伤的机制是否与肝细胞凋亡有关？方法 建立原位肝I／R模型，将肝硬化大鼠随机分为2组：A组：缺血时间（I）＝20min;B组：I＝30min;C组：正常大鼠，I＝30min，比较灌注前后各组血清AST、ALT的变化和肝细胞凋亡的百分数。结果 肝硬化大鼠肝I／R后，AST、ALT明显升高，以灌注后6h为高峰，灌注24、72h后逐渐下降。灌注6h后，B组的血清转氨酶为3组中最高（P＜0．05），说明B组肝损伤最严重。肝细胞凋亡在I／R后明显增多，以灌注后6h为高峰，随后逐渐下降，变化与转氨酶一致。灌注后6h，B、A、C组肝细胞凋亡的百分数分别为20．9％、13．5％和10．7％，B组明显高于A、C两组（P＜0．01）。再灌注72h内未见明显肝细胞坏死。结论 肝细胞凋亡是肝硬化大鼠I／R损伤肝细胞死亡的主要形式，肝细胞凋亡与肝缺血时间密切相关，肝硬化肝细胞比正常肝细胞容易发生凋亡是硬化肝对缺血敏感的重要原因。     

The CD154-CD40 T-cell co-stimulation pathway in liver ischemia and reperfusion inflammatory responses

BACKGROUND: Ischemia-reperfusion (I/R) injury is a prime antigen-independent inflammatory factor in the dysfunction of liver transplants. The precise contribution of T cells in the mechanism of I/R injury remains to be elucidated. As the CD154-CD40 co-stimulation pathway provides essential second signal in the initiation and maintenance of T-cell-dependent immune responses, this study was designed to assess the role of CD154 signaling in the pathophysiology of liver I/R injury. METHODS: A mouse model of partial 90-min warm hepatic ischemia followed by 6 hr of reperfusion was used. Three animal groups were studied: (1) wild-type (WT) mice treated with Ad-(-gal versus Ad-CD40 immunoglobulin; (2) untreated WT versus CD154 (MR1) monoclonal antibody-treated WT mice; and (3) untreated WT versus CD154 knockout mice. RESULTS: The disruption of CD154 signaling in all three animal groups ameliorated otherwise fulminant liver injury, as evidenced by depressed serum glutamic oxaloacetic transaminase levels, compared with controls. These beneficial effects were accompanied by depressed hepatic T-cell sequestration, local decrease of vascular endothelial growth factor expression, inhibition of tumor necrosis factor-(and T-helper type 1 cytokine production, and induction of antiapoptotic (Bcl-2/Bcl-xl) but depression of proapoptotic (caspase-3) proteins. CONCLUSIONS: By using in parallel a gene therapy approach, pharmacologic blockade, and genetically targeted mice, these findings document the benefits of disrupting CD154 to selectively modulate inflammatory responses in liver I/R injury. This study reinforces the key role of CD154-CD40 T-cell co-stimulation in the pathophysiology of liver I/R injury.     

Pyruvate inhibits hepatic ischemia-reperfusion injury in rats

BACKGROUND: Ischemia/reperfusion (I/R) injury is a limiting factor in liver transplantation. We have recently shown that pyruvate (PY) inhibits intestinal and renal I/R injury. This study aims to evaluate the protective effect of PY on hepatic I/R injury. METHODS: ACI rats were treated with PY, whereas control animals received placebo. Rats were killed after 60 min of partial hepatic ischemia and after 2, 6, 24, and 48 hr of reperfusion. For each time point, serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and liver biopsy specimens were obtained to evaluate morphology, DNA fragmentation, and apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling). RESULTS: The survival rate 48 hr after I/R was 83% in the control group, and 100% in the PY-treated group (P>0.05). Increased enzymatic levels and histologic findings showed increased liver damage in the untreated group compared with PY. In control rats, apoptosis was enhanced after 1 hr of ischemia and peaked after 2 hr of reperfusion, to decrease gradually 48 hr after reperfusion; in the PY group apoptosis was delayed and reduced. After 1 hr of ischemia, the number of apoptotic nuclei was significantly increased in control livers compared with normal preischemic livers, whereas the number was significantly reduced by PY. After 2 hr of reperfusion, the maximum number of apoptotic cells was observed, whereas PY significantly reduced the amount of apoptotic cells (P<0.05). Apoptosis was delayed in PY-treated livers to 6 hr after reperfusion, peaking at a significantly lower count compared with placebo-treated controls (P<0.05). CONCLUSION: These data indicate that PY has a protective effect on I/R injury of the liver.     

CD154-CD40 T-cell costimulation pathway is required in the mechanism of hepatic ischemia/reperfusion injury,and its blockade facilitates and depends on heme oxygenase-1 mediated cytoprotection

BACKGROUND: Ischemia/reperfusion (I/R) injury remains an important clinical problem that affects both early and later allograft outcome. This study was designed to analyze the role of T cells and CD154-CD40 T- cell costimulation pathway in a mouse liver I/R model. METHODS AND RESULTS: Ninety minutes of warm ischemia followed by 4 h of reperfusion in wild-type (WT) mice resulted in a significant hepatic damage, as assessed by liver function (serum alanine aminotransferase [sALT] levels), local neutrophil accumulation (myeloperoxidase activity), and histology (Suzuki's score). In contrast, T-cell deficiency (in T-cell deficient [nu/nu] mice), disruption of the CD154 signaling (in knockout [KO] mice), or its blockade in WT recipients (after MR1 monoclonal antibody [mAb] treatment), virtually prevented hepatic I/R insult. Unlike CD154-deficient T cells, adoptive transfer of WT spleen cells fully restored hepatic I/R injury in nu/nu mice. Finally, the improved hepatic function in CD154 KO recipients, WT mice treated with CD154 mAb, or nu/nu mice infused with CD154-deficient cells resulted in consistently enhanced expression of heme oxygenase-1 (HO-1), a heat-shock protein with cytoprotective functions. CONCLUSION: This study confirms the importance of T cells, and documents for the first time the role of CD154 costimulation signals in the mechanism of hepatic I/R injury. We also show that CD154 blockade-mediated cytoprotection results and depends on HO-1 overexpression. Our data provide the rationale for human trials to target CD154-CD40 costimulation in hepatic I/R injury, particularly in the transplant patient.     

Vitamin E Succinate Enhances Steatotic Liver Energy Status and Prevents Oxidative Damage Following Ischemia/Reperfusion

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.     

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.     

Mycophenolate mofetil preconditioning protects mouse liver against ischemia/reperfusion injury in wild type and toll-like receptor 4 knockout mice

BackgroundMycophenolate mofetil (MMF), an immunosuppressive drug, exerts anti-inflammatory effects on organs during ischemia/reperfusion (I/R) injury. However, the exact function of MMF in hepatic I/R injury remains largely unknown. The purpose of this study was to explore the role and potential mechanism of MMF protection in hepatic I/R injury.MethodsMale wild type (WT) and TLR4 knockout (KO) mice were injected intraperitoneally with MMF or normal saline. Animals underwent 90 min of partial hepatic ischemia, followed by 1, 6, or 24 h of reperfusion. Hepatic histology, serum amiotransferase, inflammatory cytokines, hepatocyte apoptosis, and hepatocyte autophagy were examined to assess liver injury.ResultsTreatment with MMF significantly decreased hepatic I/R injury as indicated by a reduction in serum aminotransferase levels, Suzuki scores, and the overall degree of necrosis. MMF treatment inhibited TLR4 activation dramatically. MMF administration also significantly inhibited the activation of the NF-κB pathway and the expression of pro-inflammatory cytokines. In TLR4 KO mice, MMF still exerted protection from hepatic I/R injury. MMF treatment inhibited hepatocyte apoptosis, as indicated by reduced TUNEL staining, and reduced the accumulation of cleaved caspase-3. In addition, MMF may induce autophagy and increase autophagic flux before and after hepatic reperfusion by augmenting the expression of LC3-II, P62, and Beclin-1. The induction of autophagy by MMF treatment may be related to TLR4 activation.ConclusionsOur results indicate that MMF treatment ameliorates hepatic I/R injury. The mechanism of action likely involves the ability of MMF to decrease apoptosis and the inflammatory response while inducing autophagy.     

冷/热缺血再灌注损伤对大鼠肝脏细胞能量状态、凋亡和胀亡的影响

目的研究冷/热两种条件下肝脏缺血再灌注损伤细胞能量状态与细胞死亡方式的关系,探讨减少此类损伤的措施。方法建立SD大鼠肝门阻断热缺血再灌注模型及大鼠原位肝移植冷缺血再灌注模型,测定术后缺血1h、再灌注1、12、72h肝组织能量物质变化,凋亡、胀亡和坏死细胞,观察组织病理学变化。结果热缺血期肝组织损伤重,肝脏细胞内ATP迅速下降为对照组的21%,肝脏细胞死亡方式以胀亡为主;再灌注后,ATP开始恢复,细胞死亡方式逐渐转为以凋亡为主。而冷缺血肝脏细胞缺血期肝组织损伤相对轻,ATP仅下降为对照组的68%,细胞死亡以凋亡为主;再灌注后12hATP即完全恢复,凋亡也达到顶峰,于72h逐渐下降。相关分析表明ATP、能荷与胀亡细胞数量呈显著负相关(r=-0.774,P<0.01;r=-0.789,P<0.01),与凋亡细胞数量无明显相关。结论(1)缺血再灌注发生后,以能量状态为中心环节,轻度损伤时细胞以凋亡为主,自主清除DNA受损的细胞;损伤严重时则胀亡为主,被动清除受损细胞,提示术前或术中给予能量物质可能有利于减少胀亡及炎症反应,延长肝门阻断时间。(2)单纯以凋亡或胀亡作为衡量组织功能的唯一指标值得探讨。     

P-selectin knockout mice have improved outcomes with both warm ischemia and small bowel transplantation

AIM: To analyze the role of P-selectin in intestinal ischemia and reperfusion injury (IRI) using murine models. METHODS: A model of warm IRI wherein the SMA was occluded for 100 minutes was undertaken in the following groups (10 mice per group): Group 1 (control) wild-type (WT) C57BL6, no treatment; Group 2: 0.4 mg/kg of r-PSGL1-lg 10 minutes before and after clamping; Group 3: PSGL KO mice. Survival was assessed at 7 days; the intestine was assayed for histopathology, apoptosis, myeloperoxidase (MPO), IL1, and TNF. A second model of cold IRI followed by intestinal transplantation (IT) was undertaken in the following groups (two mice per group): Group A WT --> WT: Group B PSGL KO --> WT (1-hour ischemia); Group C: PSGL KO --> WT (2 hour ischemia). Survival only was assessed. RESULTS: Survival was 50% in group 1, 90% in group 2, and 100% in group 3. Graded histopathology and crypt apoptosis demonstrated significantly less injury in groups B and C. MPO was not different between groups. IL1 and TNF were significantly reduce in groups 2 and 3. Following IT, survival was <12 hours in group A, >7 days in group B, and <72 hours in group C. CONCLUSION: This study clearly demonstrates the importance of P-selectin in warm and cold IRI in that the blockade of P-selectin using rPSGL1-lg or the absence of P-selectin using KO mice confers a survival advantage and reduction in tissue injury. The mechanism is unclear but appears to be independent of neutrophil infiltration.     

大鼠肝脏冷/热缺血再灌注损伤钠钾ATP酶、钙ATP酶及镁ATP酶与肝细胞死亡方式的研究

目的:研究肝脏冷/热血再灌注损伤条件下,钠钾ATP酶、钙ATPATP酶及镁ATP酶活性与细胞凋亡和胀亡的关系,并探讨减少此类损伤的途径.方法:建立大鼠肝门部分阻断热缺血1h再灌注模型及大鼠原位肝移植冷缺血1h再灌注模型,分别于再灌注0h、1h、12h和72h处死取材,测定肝细胞钠钾ATP酶、钙ATP酶及镁ATP酶活性.用形态学及Annexin V、PI双染法流式细胞仪定量测定早期细胞凋亡和胀亡百分数. 结果:与对照组相比,冷/热缺血1h(再灌注0h)后钠钾ATP酶、钙ATP酶及镁ATP酶活性均持续显著下降,再灌注1h达低谷.但热缺血再灌注后72h钠钾ATP酶活性恢复,钙ATP酶及镁ATP酶活性仍未恢复.冷缺血再灌注后12h钙ATP酶先恢复活性,72h钠钾ATP酶、镁ATP酶活性才恢复.细胞死亡方式:①冷缺血再灌注损伤,细胞死亡以凋亡为主,并于再灌注后12h后到顶峰.②热缺血1h胀亡细胞显著增多,再灌注1h后减少,细胞死亡方式转为以凋亡为主.结论:肝脏冷/热缺血再灌注损伤均能导致3种ATP酶活性下降,细胞内离子浓度失衡,热缺血期ATP酶活性严重下降,细胞死亡,以胀亡为主.冷缺血期ATP酶轻度下降,细胞死亡以凋亡为主.术前或术中给予能量物质,对提高钠钾ATP酶、钙ATP酶及镁ATP酶活性,减轻炎症反应,延长肝门阻断时间有利.     

Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model.

This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-3, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.     

肝脏缺血再灌注对肝癌肝内转移和生长作用的研究

目的:探讨缺血再灌注时间对肝癌肝内转移生长的作用。方法:建立70%缺血再灌注模型肝癌肝内转移小鼠模型。分为四组:假手术组,缺血15min组,缺血30 min组,缺血45 min组。四组都要在再灌注后注射肿瘤细胞,观察小鼠的生存时间,肿瘤大体观察,肿瘤组织切片HE染色计算肿瘤肝组织被替代区域(HRA)。结果:缺血30 min组和缺血45 min组肿瘤肝脏肝组织被替代区域更大,小鼠生存时间更短。结论:缺血30 min、缺血45 min组肝损伤相对更重,肝内肿瘤转移生长更快。     

白介素6基因敲除小鼠肝脏移植后的肝脏再生研究

目的 观察白介素6基因敲除(interleukin 6 knockout,IL-6 KO)小鼠原位肝移植后存活时间和肝脏再生的情况. 方法建立正常小鼠(C57BL/6 wild type,C57BL/6WT)和IL-6 KO小鼠的肝移植模型.38只小鼠分为3组:C57BL/6 WT→C57BL/6 WT肝脏移植对照组(n=10),IL-6 KO→IL-6 KO肝脏移植组(n=14)和IL-6 KO→C57BL/6 WT肝脏移植组(n=14).移植后观察小鼠肝移植物的存活情况.溴脱氧尿核苷(bromodeoxyuridine,BrdU)免疫组化检测移植肝脏的再生.结果 实验各组肝移植物的冷缺血时间均＜1 h.对照组小鼠肝移植后存活时间＞16 d.IL-6 K0→IL-6 KO组的肝移植后移植物不能存活(2 d).IL-6 KO→C57BL/6 WT组的肝移植后移植物不能存活(1.6 d).实验各组间存活时间比较,差异有统计学意义(F=190.09,P＜0.01).对照组小鼠肝脏移植后组织学检查证实肝脏组织损伤较轻,无组织坏死等改变.肝脏移植术后48 h BrdU摄取轻度增加.IL-6 KO小鼠肝移植后,组织学检查表现出片状坏死和肝细胞气球样变等改变.肝移植术后48 h免疫组化发现有极少数BrdU摄取.结论 IL-6 KO小鼠的肝移植后肝再生反应障碍,肝移植物不能存活.IL-6是肝脏再生反应中一个重要因子.     

NIM811, a Mitochondrial Permeability Transition Inhibitor, Prevents Mitochondrial Depolarization in Small-for-Size Rat Liver Grafts

ATP decreases markedly in small-for-size liver grafts. This study tested if the mitochondrial permeability transition (MPT) underlies dysfunction of small-for-size livers. Half-size livers were implanted into recipients of about twice the donor weight, resulting in quarter-size liver grafts. NIM811 (5 microM), a nonimmunosuppressive MPT inhibitor was added to the storage solutions. Mitochondrial polarization and cell death were assessed by confocal microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively. After quarter-size transplantation, alanine aminotransferase (ALT), serum bilirubin and necrosis all increased. NIM811 blocked these increases by >70%. After 38 h, BrdU labeling, a marker of cell proliferation and graft weight increase were 3% and 5%, respectively, which NIM811 increased to 30% and 42%. NIM811 also increased survival of quarter-size grafts. In sham-operated livers, hepatocytes exhibited punctate Rh123 fluorescence. By contrast, in quarter-size grafts at 18 h after implantation, mitochondria of most hepatocytes did not take up Rh123, indicating mitochondrial depolarization. Nearly all hepatocytes not taking up Rh123 continued to exclude PI at 18 h, indicating that depolarization preceded cell death. NIM811 and free radical-scavenging polyphenols strongly attenuated mitochondrial depolarization. In conclusion, mitochondria depolarized after quarter-size liver transplantation. NIM811 decreased injury and stimulated regeneration, probably by inhibiting free radical-dependent MPT onset.     

Lack of a protective effect of insulin on three reperfusion-liver injury models in rats and mice

Our objective was to investigate the potential protective effects of insulin on the liver injury induced in three ischemia and reperfusion (I/R) models. METHODS: Three I/R models were used: (1) I/R of the liver was produced in isolated, perfused rat livers; (2) in in situ I/R of the liver in rats, ischemia was induced by clamping off the hepatic artery and portal vein for 40 minutes, the flow then restored, and the liver reperfused for 90 minutes; (3) in in situ I/R of the liver in mice, ischemia was induced by clamping off the hepatic artery for 15 minutes, the flow then restored, and the liver reperfused for 45 minutes. In all three cases, blood samples collected before ischemia and after reperfusion were analyzed for sGOT. Plasma nitrate/nitrite, hydroxyl radicals, and tumor necrosis factor were also measured. In each model, a dose of insulin sufficient to induce euglycemia was administered to assess its protective effect on liver injury and inflammation. RESULTS: These I/R protocols resulted in a significant increase in sGOT and in three inflammatory parameters; nitric oxide, hydroxyl radicals, and tumor necrosis factor. Pretreatment with insulin did not attenuate the liver injury in any of the three I/R models. CONCLUSIONS: Although insulin has been reported to provide anti-inflammatory benefits by reducing oxidative and nitrosative stress and cytokine release, none of these protective effects was seen in the three I/R-induced liver injury models we tested.     

Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice

BACKGROUND: Ischemia/reperfusion injury (I/R injury) of the liver remains a significant problem during liver surgery and transplantation. I/R injury is associated with liver apoptosis, which is mediated by death receptors such as Fas and tumor necrosis factor alpha (TNF-alpha), and/or mitochondrial dysfunction induced by cellular stress. Caspase-8 is presumed to be the apex of the death-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" proteases in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8 and caspase-3 siRNA in a murine model of liver I/R injury. METHODS: In C57BL/6 mice, 45% or 70% of the liver mass was clamped for 90 minutes. For survival analysis, total hepatic ischemia was induced for 45 minutes. In vivo delivery of siRNA was performed via the portal vein by high-volume injection (0.5 nmol of siRNA in 1 mL containing 10% lipiodol) 60 minutes before ischemia. As a control, animals received either vehicle or non-sense siRNA (siRNA-scrambled). RESULTS: Liver uptake of siRNA was analyzed in transgenic mice who express beta-galactosidase (beta-gal) (C57BL/6J-TgN(MTn-LacZ)204Bri) after administration of siRNA-LacZ. A 3- to 4-fold decrease in beta-gal activity was accomplished at 0.5 nmol. No significant change in beta-gal activity was demonstrated in mice receiving non-sense siRNA. Immunohistochemical studies found that 60% of the liver cells efficiently took up siRNA. Significant reduction in serum aspartate transaminase was found in animals treated with siRNA caspase-8 or caspase-3 compared with siRNA-scrambed or vehicle-treated controls. More than a 60% reduction in caspase-8 and caspase-3 gene expression and activities was accomplished after siRNA administration. Animals treated with siRNA presented lower infiltration of polymorphonuclear leukocytes and better preservation of the liver architecture compared with controls. All of the control mice subjected to total liver ischemia died within 5 days. In contrast, 30% of the animals given siRNA caspase-8 and 50% of those treated with siRNA caspase-3 survived indefinitely (>30 days). CONCLUSIONS: Small interfering RNA targeted to caspase-8 and caspase-3 provided significant protection against I/R injury to the liver. This approach could be therapeutic in liver transplantation and other conditions associated with I/R injury to the liver.     

Attenuation of ischemia and reperfusion injury of canine livers by inhibition of type II phospholipase A2 with LY329722

BACKGROUND: Membrane phospholipid breakdown, caused by ischemia and reperfusion (I/R) of the liver, releases free fatty acids including arachidonic acids and lysophospholipids, which serve as precursors of various inflammatory lipid derivatives. Phospholipase A2 (PLA2) is a key enzyme that initiates this reaction. In this study, we tested our hypothesis that a type II PLA2 inhibitor, LY329722, could attenuate hepatic I/R injury caused by a 2-hr total hepatic vascular exclusion (THVE) in dogs. METHODS: Eighteen beagle dogs, subjected to a 2-hr THVE, were divided into three groups. Group 1 (n=6) was untreated and served as a control group. LY329722 was administered to animals in group 2 (n=6) intravenously (0.2 mg x kg(-1) x hr(-1)) for 60 min before ischemia, and to animals in group 3 (n=6) for 60 min starting 15 min before reperfusion (0.2 mg x kg(-1) x hr(-1)). Animal survival, systemic and splanchnic hemodynamics, hepatic tissue blood flow, liver functions, energy metabolism, hepatic venous thromboxane B2 and endothelin-1 levels, phospholipid levels and tumor necrosis factor-a mRNA expression in liver tissue, and histopathologic findings were evaluated. RESULTS: Two-week animal survival was 33% (two of six) in group 1, and 100% (six of six) in groups 2 and 3. LY329722 improved systemic and splanchnic hemodynamics, hepatic tissue blood flow, and energy metabolism, reduced liver enzyme, thromboxane B2, and endothelin-1 release, prevented hepatic phospholipid degradation and tumor necrosis factor-alpha mRNA expression, and lessened histopathologic damage and the number of neutrophil infiltrating into the liver tissue. CONCLUSION: The present study demonstrated that a type II PLA2 inhibitor, LY329722, attenuated hepatic I/R injury caused by a 2-hr THVE model in dogs.     

Murine hindlimb reperfusion injury can be initiated by a self-reactive monoclonal IgM

BACKGROUND: Murine hindlimb reperfusion injury (I/R), is initiated by activation of the classical pathway of complement. Complement receptor-2 knockout mice (Cr2-/-) are protected from I/R injury due to defective B-1 cells with a resulting deficient natural immunoglobulin M (IgM) repertoire. Cr2-/- and wild type (WT) mice were studied to isolate the antibody or antibodies responsible for initiation of I/R. METHODS: IgM-secreting B-1 cell clones were produced with hybridoma technology from WT cells. Of 21 clones tested in murine I/R models, only 1 clone, CM22, was found to restore injury in protected mice. Cr2-/- mice reconstituted with IgM from individual clones, WT serum, or saline were subjected to 2 hours hindlimb ischemia and 3 hours reperfusion and compared with WT. RESULTS: Muscle injury in Cr2-/- mice reconstituted with CM22 was similar to injury in WT mice reconstituted with saline and Cr2-/- mice reconstituted with WT serum. This injury was 137% greater (P < .05) than in both Cr2-/- mice reconstituted with saline and those reconstituted with a different IgM clone, CM31. IgM and C3 deposition was found only on injured muscle of WT mice or Cr2-/- mice reconstituted with CM22 or WT serum. CONCLUSION: A single clone of self-reactive IgM, CM22, can initiate complement-dependent I/R injury.