缺血再灌注损伤与细胞凋亡

目的 研究缺血再灌注过程中细胞凋亡现象的发生及其相关机理。方法 采用文献回顾的方法对缺血再灌注期间细胞凋亡现象的发生及其相关机理进行综述。结果 多种脏器、组织历经缺血再灌注后均发现有细胞凋亡现象，缺血再灌注期间影响细胞凋亡的因素有多种，如缺血、缺氧、氧自由基、细胞内钙超载、多种细胞因子等，而细胞凋亡的调控与多种基因的调控有关，如bcl-2家族、caspase家族及核因子NF－κB等。结论 细胞凋亡是脏器、组织在缺血再灌注期间的一种普遍现象，它受多种因素的影响和调节。     

Bupropion Reduces the Inflammatory Response and Intestinal Injury Due to Ischemia-Reperfusion

Intestinal ischemia-reperfusion (I/R) causes severe organ failure and intense inflammatory responses, which are mediated in part by the cytokine tumor necrosis factor-alpha (TNF-alpha). Bupropion is an antidepressant known to inhibit TNF-alpha production. We sought to examine the protective effects of bupropion on intestinal I/R injury in 15 male Sprague-Dawley rats that were randomized to sham surgery, 45 minutes of intestinal ischemia followed by 180 minutes reperfusion, or bupropion (100 mg/kg) before the intestinal I/R injury. To evaluate the systemic inflammatory response induced by intestinal I/R, we measured serum levels of TNF-alpha, interleukins-1 and -6, lipid peroxidation, and transaminases. Histologic analysis evaluated intestinal injury using the Chiu muscosal injury score. After I/R, Chiu score in control animals was 3.6 ± 1.2 vs 2.6 ± 0.53 in animals that received bupropion (P < .05). Bupropion pretreatment reduced intestinal. I/R injury and blunted serum elevations of TNF-alpha (0.96 ± 1.1 ng/mL vs 0.09 ± 0.06 ng/mL, P < .05) and interleukin-1 (0.53 ± 0.24 ng/mL vs 0.2 ± 0.11 ng/mL, P < .05). Bupropion in reduced intestinal I/R injury through immunomodulatory machanisms that involve inflammatory cytokines such as TNF-alpha.     

Lower Limb Ischemia-Reperfusion Injury Triggers a Systemic Inflammatory Response and Multiple Organ Dysfunction

Restoration of blood flow to an acutely ischemic lower limb may, paradoxically, result in systemic complications and unexpected mortality. We investigated the effect of acute ischemia-perfusion of the lower limb on cytokine production and end organ function. Plasma concentrations of tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were determined in five groups of male Wistar rats: control, 3 hours of bilateral hind limb ischemia alone, and 3 hours of bilateral hind limb ischemia followed by 1 hour, 2 hours, or 3 hours of reperfusion, respectively. In a second experiment, the effect of lower limb ischemia-reperfusion on remote organs (lung, liver, and kidney) was assessed biochemically and histologically. There was a significant increase in plasma concentrations of TNF-a in plasma of animals subjected to 3 hours of bilateral hind limb ischemia followed by 1 hour of reperfusion, 40.1 +/- 4.4 pg/ml, when compared with controls, 22.6 +/- 4.4 pg/ml, or animals in the ischemia-alone group, 16.3 +/- 5.2 (p <0.05). Plasma concentration of IL-6 increased progressively and significantly in animals subjected to bilateral hind limb ischemia followed by 1 hour of reperfusion, 720 +/- 107 pg/ml; 2 hours of reperfusion, 1987 +/- 489 pg/ml; or 3 hours of reperfusion, 6284 +/- 1244 (p <0.0001), compared with controls, 104 +/- 43 pg/ml; or animals in the ischemia-alone group, 140 +/- 55 pg/ml. In the study comparing portal and systemic concentrations of IL-6, systemic concentrations of IL-6, 967 +/- 184 pg/ml were significantly higher than those in the portal circulation 577 +/- 127 pg/ml (p <0.05). There was a significant increase in plasma concentrations of urea, creatinine, aspartate transaminase, alanine transaminase, and lactic dehydrogenase in reperfused animals compared with controls (p <0.001). Morbidity and mortality following reperfusion of the acutely ischemic limb may be a manifestation of multiple organ dysfunction caused by a systemic inflammatory response triggered by reperfusion of the ischemic extremities.     

缺血后处理对鼠在体肺缺血-再灌注损伤炎症反应作用的实验研究

目的 探讨缺血后处理对大鼠在体肺缺血-再灌注损伤中炎症反应的影响。 方法 将40只SD大鼠随机分成假手术组 （S组）、缺血-再灌注30 min组 （I/R-30组）、缺血-再灌注120 min组 （I/R-120组）、缺血后处理30 min组 （IPO-30） 和缺血后处理120 min组 （IPO-120）,每组8只。S组完成左侧开胸手术操作,肺门过阻断带,但肺门未阻断;I/R组拉紧肺门阻断带阻断左肺门,造成左肺缺血1 h,然后松开阻断带再灌注30 min和120 min （即I/R-30组和I/R-120组）;IPO组在1 h的缺血之后再灌注开始前先进行10 s的缺血及10 s的再灌注（共3个循环,持续1 min）,然后是与I/R组同样的30 min和120 min的长时间持续灌注（即IPO-30组和IPO-120组）。测定各组肺组织中白细胞介素10（IL-10） 及血浆可溶性细胞间粘附分子1 （sICAM-1） 的水平。观察肺组织的病理形态变化并进行弥漫性肺泡损伤（DAD） 评分。 结果 I/R-30组和I/R-120组血浆中sICAM-1水平比S组显著升高［（2.140±0.250） μg/L vs. （0.944±0.188） μg/L,P=0.003; （2.191±0.230） μg/L vs. （0.944±0.188） μg/L,P=0.003］,肺组织中IL-10水平亦明显高于S组［（15.922±0.606） pg/mg pro vs. （7.261±0.877） pg/mg pro,P=0.037; （17.421±1.232） pg/mg pro vs. （7.261±0.877） pg/mg pro,P=0.042］,组织损伤明显。缺血后处理干预后,IPO-30组和IPO-120组血浆sICAM-1水平与相应I/R组各时间点相比显著降低 ［（1.501±0.188） μg/L vs. （2.140±0.250） μg/L,P=0.038; （1.350±0.295） μg/L vs. （2.191±0.230） μg/L,P=0.005］,而IL-10水平显著升高［（20.950±1.673） pg/mg pro vs. （15.922±0.606） pg/mg pro,P=0.008; （25.334±1.173） pg/mg pro vs. （17.421±1.232） pg/mg pro,P=0.006］,DAD评分显著降低［6.8±1.4 vs. 11.5±1.9,P=0.007;7.5±1.6 vs. 13.2±1.7,P=0.005］,肺组织损伤较I/R组显著减轻。 结论 缺血后处理可能通过抑制炎症反应从而减轻肺缺血-再灌注损伤。     

Recombinant Human Erythropoietin Pretreatment Attenuates Heart Ischemia-Reperfusion Injury in Rats by Suppressing the Systemic Inflammatory Response

Background

Ischemia-reperfusion (I/R) injury may influence graft function after transplantation. Erythropoietin (EPO) attenuates I/R injury in various animal organs such as intestine, brain, and kidney.

Objective

To evaluate the effects of pretreatment with recombinant human EPO (rhEPO) on I/R-induced heart injury.

Materials and Methods

A rat model of I/R injury was established by ligating the left descending coronary artery for 30 minutes, followed by reperfusion for 4 hours. Fifty Sprague-Dawley rats were divided into 5 groups: sham operation; I/R; I/R+rhEPO, 100 U/kg; I/R+rhEPO, 1000 U/kg; and I/R+rhEPO, 5000 U/kg. Electrocardiograms were assessed continuously to note arrhythmia caused by reperfusion. Serum concentrations of interleukin (IL)-6 and IL-8, and tumor necrosis factor-α were measured at 2 and 4 hours after reperfusion.

Results

The rhEPO-treated animals exhibited dosage-dependent significant reduction in the incidence of ventricular arrhythmia caused by reperfusion, and markedly decreased serum concentrations of IL-6, IL-8, and tumor necrosis factor-α (P < .05) compared with the I/R group (P < .05).

Conclusion

The rhEPO attenuates myocardial I/R injury in rats, at least in part related to inhibition of the system inflammatory response.     

Renal Ischemia-Reperfusion Injury Amplifies the Humoral Immune Response

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24–96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG₁ compared with sham-treated controls. This amplified IgG₁ response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.Delayed graft function (DGF), defined as the need for dialysis in the first 7 days after transplantation surgery,¹ is associated with an increased risk of acute rejection.^2,3 The increased risk of rejection is seen with both cadaveric³ and live donor⁴ organs, and also, it is associated with increased recipient mortality.^5,6 Furthermore, long-term survival is worse in DGF kidney grafts, and there is an increasing recognition that ongoing inflammation drives interstitial fibrosis/tubular atrophy (formerly known as chronic allograft nephropathy)^7–9 and may limit average duration of transplant survival.Although it is well known that a host of systemic inflammatory diseases cause renal injury, far less is known about the effect of AKI on the immune system. Animal and human data seem to be conflicting, indicating that AKI both is an intensely inflammatory event^10–12 and may also have immunosuppressive effects.¹³ Ischemia-reperfusion injury (IRI) of native kidneys may also perturb systemic immune responses. AKI is associated with acute respiratory distress syndrome and synergistically increases patient mortality because of sepsis and multiorgan dysfunction syndrome.¹⁴With the increasing incidence of AKI,^15,16 it is important to understand the effects of AKI on immune function. Long-lasting immune lymphocyte activation has been shown after IRI.¹⁷ In the specific setting of renal transplantation, stubbornly high rates of late graft failure are now thought to involve persistent subclinical immune activity,^18,19 and basic science work has shown a variety of immune factors that are active in the failing allograft, including T cells, selectins, integrins, and chemokine receptors such as chemokine receptor-2, chemokine receptor-7, and CXC chemokine receptor-4.⁸ There is an increasing awareness that ischemic kidney damage causes local inflammation in the kidney. In the case of renal allografts that suffer ischemic damage, it may be a pathologic process ultimately leading to increased antibody-mediated rejection.These observations led us to hypothesize that IRI amplifies the humoral immune response to a newly presented antigen. To test this hypothesis, we subjected mice to bilateral renal IRI and then immunized them with nitrophenol keyhole limpet hemocyanin (NP-KLH), a large T cell-dependent antigen that is commonly used in humoral immunity experiments. We show the effect of IRI on antibody levels after immunization and explore mechanisms for the observed phenomena.     

Glucose Intolerance Modifies the Inflammatory Response After Intestinal Ischemia-Reperfusion

Streptozotocin administration in newborn rats (nSTZ-rats) leads to adults with mild insulin deficiency and normoglycemia, and is accepted as a model of type 2 diabetes. We examined possible differences in the production of inflammatory mediators between healthy and nSTZ-rats after ischemia-reperfusion (I-R). Two-month-old control and nSTZ-rats were randomly separated into control and intestinal I-R groups. After reperfusion, samples were obtained from the portal vein (PV) infrahepatic cava vein (ICV), suprahepatic cava vein (SCV), jejunal wall, and pancreas. Nitric oxide (NO), lipid hydroperoxides (LPO), tumor necrosis factor alpha (TNF-α), 60 kDa receptor (sTNF-R1), 80 kDa (sTNF-R2), and intercellular adhesion molecule-1 (ICAM-1), were determined. After I-R, nSTZ-rats showed increased plasma concentrations of LPO, NO, ICAM-1 (0.5141 ± 0.083 vs 0.024 ± 0.003, ICV; 0.574 ± 0.075 vs 0.023 ± 0.003, SCV; 0.528 ± 0.067 vs 0.027 ± 0.003 PV; ng/ml), TNF-α (42.4 ± 5.7 ICV, 248.4 ± 28.2 SCV, and 33.6 ± 4.0 PV. In n STZ-rats, vs 4.36 ± 0.57, 4.74 ± 0.77, and 3.16 ± 0.32, respectively, in control rats; pg/ml), and sTNF-R1. Both TNF-α and NO plasma levels were higher in SCV than in ICV and PV after I-R. In addition, after I-R, jejunal wall of nSTZ-rats showed an increase of TNF-α IL-1, and IL-10 levels. A pre-existing state of glucose intolerance intensifies the inflammatory response after intestinal I-R.     

Novel Approaches to Preventing Ischemia-Reperfusion Injury During Liver Transplantation

Ischemia-reperfusion injury (IRI) results in profound allograft damage during liver transplantation. The process of IRI results in adenosine triphosphatase (ATP) depletion, the production of reactive oxygen species, and progressive tissue destruction. This injury process is accelerated on reperfusion in the recipient. Over the last decade an increasing body of literature has identified a complex interplay of molecular and cellular pathways responsible for causing IRI. This article summarizes recent developments, drawing on preclinical and clinical studies, focusing on how the detrimental effects of IRI can be prevented in liver transplantation. We present a balanced overview on how machine preservation technologies, the coagulation system, antioxidants, cytoprotective agents, cytokines, preservation solutions, and the innate and adaptive immune system can be targeted to prevent IRI in liver transplantation.     

Activation of GPR81 Aggravates Remote Organ Injury During Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion injury (HIRI) is a serious situation with high morbidity and mortality, which is usually accompanied with hyperlactatemia due to impaired lactate clearance in liver. G-protein-coupled receptor 81 (GPR81) has recently been identified as the bioactive receptor of lactate. GPR81 is profoundly involved in the modulation of metabolism and inflammation, but its significance in HIRI remains unclear. The present study investigated the potential roles of GPR81 in HIRI by using the GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA). The results indicated that treatment with CHBA had no obvious effects on HIRI-induced histologic abnormalities and elevation of serum aspartate aminotransferase, alanine aminotransferase. However, CHBA significantly upregulated the serum level of tumor necrosis factor alpha and interleukin-6 in mice with HIRI. Administration of CHBA also exacerbated HIRI-induced histologic lesions in lung, increased the level of myeloperoxidase in lung tissue and the protein concentration in bronchoalveolar lavage fluid. In addition, the serum levels of brain natriuretic peptide and creatinine also increased in CHBA-treated mice. The results indicate that activation of GPR81 might aggravate HIRI-induced remote organ injury and result in serious outcomes.     

IL-6 May Modulate the Skeletal Response to Glucocorticoids During Exacerbations of Inflammatory Bowel Disease

Whether inflammatory cytokines affect the skeletal response to glucocorticoid (GC) treatment is unclear. Our objectives were to (1) identify the cytokine(s) elevated during exacerbations of inflammatory bowel disease (IBD); (2) determine whether the cytokine(s) identified in this way is related to systemic GC sensitivity; and (3) examine whether cytokines and/or measures of GC sensitivity are related to changes in bone formation or resorption following GC therapy. We designed a combined cross-sectional and prospective study, including patients with active (n = 31) and inactive (n = 34) IBD as well as controls (n = 29). We assessed circulating concentrations of cytokines, PINP and βCTX, as well as GC sensitivity in peripheral blood mononuclear cells. IL-6 was the only cytokine increased in active IBD, 2.35 (2.63) versus 1.64 (1.21) versus 1.31 (2.79) pg/μl active IBD, inactive IBD, and controls, respectively (median [interquartile range]) (P = 0.03, ANOVA). IL-6 was positively related to magnitude of GC sensitivity (beta = 0.02, 95% CI 0.008–0.04, P = 0.005). Following treatment with GC in active IBD, PINP decreased (P < 0.001), whereas βCTX showed no significant change (P = 0.2). Subsequently, multiple regression analyses revealed that plasma IL-6 concentrations were inversely related to the extent of PINP suppression following GC (beta = 3.3, 95% CI 0.2–6.4, P = 0.04, adjusted for baseline PINP and duration of GC treatment), while no association was observed with GC sensitivity. In conclusion, IL-6 is elevated in active IBD and may protect against GC-induced suppression of bone formation via a mechanism which appears to be independent of systemic GC sensitivity.     

骨髓间充质干细胞对缺血再灌注损伤肾小管上皮细胞增殖和凋亡的影响

目的：探讨外源性骨髓间充质干细胞（MSCs）移植对缺血再灌注损伤（I/R）后肾小管上皮细胞增殖和凋亡的影响。方法：将雄性SD大鼠MSCs用DAPI标记后注入受体雌性SD大鼠体内。30只受体大鼠随机分为3组：假手术对照组（C组）、MSCs＋I/R组（M组）、DMEM-F12＋I/R组（D组）,每组10只。7d后观察肾功能,肾脏病理改变,采用原位末端标记法检测细胞凋亡指数,免疫组化法检测增殖细胞核抗原（PCNA）的表达,并观察DAPI标记的MSCs在受体大鼠肾脏的分布情况。结果：I/R后第7天,M组在肾功能、肾脏病理改变上,均明显好于D组;肾组织内PCNA＋细胞数和凋亡指数均低于D组。I/R后7d内未发现MSCs定位于肾组织中。结论：外源性MSCs可以促进I/R损伤后肾小管上皮细胞的增殖和减少细胞凋亡,从而有利于肾小管损伤的早期恢复。     

Mortality following Lower Limb Ischemia-Reperfusion: A Systemic Inflammatory Response?

n = 10); and (2) animals subjected to 3 hours of bilateral hind limb ischemia followed by reperfusion ( n = 10). Both groups were observed under standard conditions for 4 days. In a second experiment three groups of animals were studied: (I) control ( n = 12); (II) 3 hours of bilateral hind limb ischemia alone ( n = 12); and (III) 3 hours of bilateral hind limb ischemia followed by 2 hours of reperfusion ( n = 12). Animals subjected to bilateral hind limb ischemia followed by reperfusion had a significantly higher mortality rate (70%) than controls (0%) ( p < 0.005). Morphometric assessment of the small bowel showed a significant decrease in mean mucosal thickness in the ischemia-reperfusion group compared with that in the group of controls and the ischemia-alone group ( p < 0.05). Bilateral hind limb ischemia followed by reperfusion was associated with significantly increased plasma concentrations of endotoxin ( p < 0.05) and interleukin-6 ( p < 0.0001) compared with that of controls and ischemia alone. These results indicate that reperfusion of the acutely ischemic lower limb is accompanied by structural changes in the gut mucosa associated with increased systemic endotoxin concentrations and cytokine activation. Mortality following reperfusion of the acutely ischemic limb may be related to a systemic inflammatory response triggered by endotoxin of gut origin.     

Hepatocellular Glycogen in Alleviation of Liver Ischemia-Reperfusion Injury During Partial Hepatectomy

Background Temporary occlusion of liver blood supply for complex liver operation is common in liver surgery. However, hepatic vascular occlusion will undoubtedly impair liver function. This study was designed to elucidate the effect of hepatocellular glycogen in alleviation of liver ischemia-reperfusion injury during hepatic vascular occlusion for partial hepatectomy. Methods Fifty-seven patients were randomly divided into an experimental group (n = 29) and a control group (n = 28). In the experimental group, patients were given high-concentration glucose intravenously during 24 h before the operation. The hepatic lesion was resected after portal triad clamping in the two groups. Noncancer liver tissue was biopsied to measure hepatic tissue ATP content and change of malondialdehyde (MDA) and superoxide dismutase (SOD). Liver function of all patients was assessed by using an automatic biochemical analysis apparatus before the operation and the first and fifth days after operation. Results The mean hepatic vascular occlusion time in the experimental group was 19.21 ± 4.54 min and in the control group it was 21.04 ± 5.11 min. Hepatic tissue ATP content of the experimental group was significantly higher than that of the control group at the end of hepatic vascular occlusion (2.15 ± 0.39 μmol/g wet tissue vs. 1.33 ± 0.44, p < 0.01) and at the point of 1-h reperfusion (2.19 ± 0.29 μmol/g wet tissue vs. 1.57 ± 0.35, p < 0.01). There was significant difference in SOD activity between the two groups at the end of hepatic vascular occlusion (130.69 ± 30.49 NU/mg pr vs. 97.83 ± 26.23, p < 0.01) and at the point of 1-h reperfusion (139.55 ± 39.88 NU/mg pr vs. 114.74 ± 25.93, p < 0.01). Significant difference was shown in MDA content between the two groups at the end of hepatic vascular occlusion (3.02 ± 0.30 nmol/mg pr vs. 3.99 ± 0.49, p < 0.01) and at the point of 1-h reperfusion (3.81 ± 0.69 nmol/mg pr vs. 5.75 ± 1.17, p < 0.01). In addition, the liver function of the experimental group was significantly better than that of the control group the first and fifth days after the operation (p < 0.01). Conclusions Abundant intracellular glycogen may reduce liver ischemia-reperfusion injury caused by hepatic vascular occlusion. It is beneficial to give a large amount of glucose before a complex liver operation during which temporary occlusion of hepatic blood flow is necessary.     

Hyperoside Attenuates Hepatic Ischemia-Reperfusion Injury by Suppressing Oxidative Stress and Inhibiting Apoptosis in Rats

PurposeHepatic ischemia-reperfusion (IR) injury is a serious complication of many clinical conditions, which may lead to liver or multiple organ failure. Hyperoside, a flavonoid compound, has been reported to protect against myocardial and cerebral injury induced by IR. This study aimed to investigate the protective effects of hyperoside on hepatic IR injury in rats.MethodsUsing the 70% hepatic IR injury model, we divided 32 male Wistar rats into 4 groups (n = 8): sham-operated, IR+saline (saline/p.o.), IR+vehicle (carboxy methyl cellulose/p.o.), and IR+hyperoside (50 mg/kg/d/p.o.). At 24 hours after reperfusion, blood and liver tissue were collected. The effects of hyperoside on hepatic IR injury were assessed through tests of serum transaminase, hepatic histopathology, and measurement of markers of oxidative stress and apoptosis.ResultsPretreatment with hyperoside protected the liver from IR injury by a reduction in serum aspartate aminotransferase/alanine aminotransferase levels and a decrease in the severity of histologic changes. Hyperoside treatment also decreased the activity of malondialdehyde, increased the activities of superoxide dismutase and glutathione peroxidase, up-regulated the expression of heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1, and reduced the apoptotic index after IR injury. A decrease in the expression of caspase–3 and an increase in the ratio of B cell lymphoma 2 to B cell lymphoma 2–associated X also were observed.ConclusionHyperoside has a protective effect on hepatic IR injury in rats, which may be due to its antioxidant and antiapoptotic properties.     

P-选凝素与心肌缺血-再灌注损伤

研究心肌缺血 -再灌注损伤中 P-选凝素 ( P- selectin)的重要作用。P- selectin是一种糖蛋白黏附因子 ,存在于内皮细胞和血小板 ,并介导血小板、内皮细胞和多形核白细胞 ( PMNs)等之间的相互作用 ,且与一氧化氮有着密切的关系 ,形成了许多复杂的炎症病理过程 ,在心肌缺血 -再灌注损伤中起到了关键的作用。特别是 P- selectin与晚期再灌注损伤、血小板及心肌损伤中治疗作用、最新的 P- selectin基因缺陷小鼠和糖尿病小鼠等的心肌缺血 -再灌注损伤中表现的深入研究 ,使其在缺血 -再灌注损伤中的重要性和复杂性显得更加突出。     

缺血-再灌注损伤对大鼠急性胰腺炎胰腺细胞凋亡的影响

目的探讨缺血一再灌注(I／R)损伤对大鼠急性胰腺炎(AP)胰腺细胞凋亡的影响。方法将SD大鼠54只按编号法随机分为对照组(n=6)、胰腺炎组(n=24)和I／R损伤组(n=24)。经胆胰管逆行加压注入3％牛磺胆酸钠建立大鼠AP模型，在此基础上，I／R损伤组通过暂时阻断脾下动脉造成局部胰腺I／R模型，对照组于术后lh，其余两组于术后1h、3h、6h和12h采取断颈方法分批处死动物，应用末端脱氧核苷酸转换酶(TdT)介导的原位末端标记(TUNEL)法检测缺血一再灌注区胰腺细胞凋亡。并观察其病理改变。结果胰腺炎组大鼠术后1h、3h胰腺组织仅为充血、水肿性改变，6h出现出血、坏死性改变；而1／R损伤组大鼠术后1h缺血一再灌注区胰腺呈现出血、坏死性改变，病变持续加重；AP后胰腺凋亡细胞明显增多，I／R损伤组和胰腺炎组的凋亡细胞高峰值分别在术后3h和6h；I／R损伤组术后1h、3h缺血一再灌注区胰腺凋亡细胞显著高于相应时相的胰腺炎组(P＜0．01，P＜0．05)．而6h、12h明显低于胰腺炎组(P＜O．05，P＜O．01)。结论I／R损伤在促发胰腺炎从水肿型向出血坏死型转化过程中，同时诱导胰腺细胞凋亡，细胞凋亡可能是阻止AP病变加重的一个有利反应。     

The Effect of K-ATP Channel Blockage During Erythropoietin Treatment in Renal Ischemia-Reperfusion Injury

ATP dependent K channels (K-ATP) take part in the Erythropoietin (EPO) induced cardioprotection but these channel activations have role in cytoprotective role of EPO in the renal ischemia reperfusion (IR) damage is still unknown. For this purpose rats were pretreated with EPO (500 IU/kg) and/or K-ATP channel blocker glibenclamide (40mM/kg) i.p. before bilateral renal IR damage. Renal tissues were used for histological examination and measurement of caspase-3 and TNF-α levels. Renal functions were evaluated by glomerular filtration rate (GFR) fractional excretion of sodium (FENa) and potassium (FEK). Renal TNF-α and caspase-3 levels were decreased in both glibenclamide and EPO-treated IR rats compared to untreated rats. The protection afforded by the pretreatment with EPO alone was greater than that of administering glibenclamide alone. Application of glibenclamide at the same time partly abolished the cytoprotective effect of EPO treatment. K-ATP mediated cytoprotection is not the main mechanism of protective effect of EPO.     

Increasing Resistance of Tubular Epithelial Cells to Apoptosis by shRNA Therapy Ameliorates Renal Ischemia-Reperfusion Injury

Renal tubular epithelial cells (TEC) die by apoptosis or necrosis in renal ischemia-reperfusion injury (IRI). Fas/Fas ligand-dependent fratricide is critical in TEC apoptosis, and Fas promotes renal IRI. Therefore, targeting Fas or caspase-8 may have therapeutic potential for renal injury in kidney transplant or failure. RNA silencing by short hairpin RNA (shRNA) is a novel strategy to down-regulate protein expression. Using this approach, silencing of Fas or caspase-8 by shRNA to prevent TEC apoptosis and IRI was evaluated. IRI was induced by renal artery clamping for 45 or 60 min at 32 degrees C in uninephrectomized C57BL/6 mice. Here, we showed that Fas or pro-caspase-8 expression was significantly knocked down in TEC by stable expression of shRNA, resulting in resistance to apoptosis induced by superoxide, IFN-gamma/TNF-alpha and anti-Fas antibody. Inferior vena cava delivery of pHEX-small interfering RNA targeting Fas or pro-caspase-8 resulted in protection of kidney from IRI, indicated by reduction of renal tubular injury (necrosis and apoptosis) and serum creatinine or blood urea nitrogen. Our data suggest that shRNA-based therapy targeting Fas and caspase-8 in renal cells can lead to protection of kidney from IRI. Attenuation of pro-apoptotic proteins using genetic manipulation strategies such as shRNA might represent a novel strategy to promote kidney allograft survival from rejection or failure.