虎杖甙对肠缺血再灌流时肠外器官的保护作用

本实验探讨了家兔肠缺血再灌流损伤时肠外器官损害的发生机制，观察了虎杖甙对器官的保护作用。结果显示，与肠缺血再灌流损伤组相比较，虎杖甙处理组动物肺通透指数上升的幅度显著降低。     

Triggers of inflammation after renal ischemia/reperfusion

Renal ischemia/reperfusion (I/R) is a common cause of acute renal failure (ARF). Ischemic ARF is associated with tubulointerstitial inflammation, and studies using animal models have demonstrated that the inflammatory response to I/R exacerbates the resultant renal injury. Ischemic ARF involves complement activation, the generation of cytokines and chemokines within the kidney, and infiltration of the kidney by leukocytes. Recent work has revealed some of the events and signals that trigger the inflammatory response to aseptic, hypoxic injury of the kidney. In many ways, the inflammatory reaction to this injury resembles that seen during ascending urinary infection, and it may represent a general response of the tubular epithelial cells (TECs) to stress or injury. A greater understanding of the signals that trigger the inflammatory response may permit the development of effective therapies to ameliorate ischemic ARF.     

Vacuole formation and endothelial damage in microvessels after ischemia reperfusion

Vacuole formation and endothelial damage in microvessels after ischemia reperfusion     

Cryotherapy reduces skeletal muscle damage after ischemia/reperfusion in rats

The aim of this study was to analyze the effects of cryotherapy on the biochemical and morphological changes in ischemic and reperfused (I/R) gastrocnemius muscle of rats. Forty male Wistar rats were divided into control and I/R groups, and divided based on whether or not the rats were submitted to cryotherapy. Following the reperfusion period, biochemical and morphological analyses were performed. Following cryotherapy, a reduction in thiobarbituric acid‐reactive substances and dichlorofluorescein oxidation levels were observed in I/R muscle. Cryotherapy in I/R muscle also minimized effects such as decreased cellular viability, levels of non‐protein thiols and calcium ATPase activity as well as increased catalase activity. Cryotherapy also limited mitochondrial dysfunction and decreased the presence of neutrophils in I/R muscle, an effect that was corroborated by reduced myeloperoxidase activity in I/R muscle treated with cryotherapy. The effects of cryotherapy are associated with a reduction in the intensity of the inflammatory response and also with a decrease in mitochondrial dysfunction.     

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion.

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.     

A distinct subset of HLA-DR-regulatory T cells is involved in the induction of preterm labor during pregnancy and in the induction of organ rejection after transplantation

Regulatory T cells (Tregs) are known to suppress alloimmune responses during pregnancy and post organ transplantation. We demonstrate that a distinct subset of FoxP3⁺DR⁺-Tregs among the total CD4⁺CD127^low+/−CD25⁺-Treg cell pool is critically involved in preterm labor induction and kidney transplant rejection as well. Compared to healthy pregnancies and non-rejecting kidney recipients, we found that the percentage of the FoxP3⁺DR⁺-Treg subset was not reduced, but that the level of HLA-DR expression of such Tregs was strongly diminished in preterm laboring women and in patients with acute renal allograft rejection. In addition, both patient collectives showed a significantly reduced suppressive activity of their circulating CD4⁺CD127^low+/−CD25⁺-Treg cell pool. Our findings propose that the FoxP3⁺DR⁺-Treg subset may be decisively responsible for the suppressive activity of the total CD4⁺CD127^low+/−CD25⁺-Treg cell pool and that the immunologic mechanisms leading to preterm labor necessitating preterm delivery may be similar to those leading to allograft rejection after transplantation.     

Hepatocyte growth factor ameliorates renal hemodynamic disorder after ischemia/reperfusion

Ischemic injury of the transplanted kidney is one of the causes of reduced graft survival. The purpose of the present experiment was to examine whether hepatocyte growth factor (HGF) would improve acute renal hemodynamic recovery immediately after cold ischemia. Addition of HGF to the preservation solution during 3 h cold ischemia of dog kidney accelerated both recovery of renal blood flow and glomerular filtration rate (GFR). It is suggested that HGF may be useful for preservation of excised kidney for transplantation. As intrarenal arterial infusion of HGF in normal dog kidney had no effects on renal hemodynamics, mechanisms other than direct vasodilator action of HGF appear to be operating in the protection.     

异丙酚减轻大鼠脑缺血再灌注损伤

目的探讨异丙酚对脑缺血再灌注损伤的作用及其可能的机制,为临床用药提供实验依据。方法 将大鼠随机均分为假手术组、异丙酚预处理组、大脑缺血-再灌注组。用Longa法成功制备,左侧大脑中动脉闭塞(MCAO)模型,于再灌注24 h后,取大脑切片行2,3,5-氯化三苯基四氮唑染色,测量并计算脑梗死容积百分比。流式细胞仪检测大鼠脑缺血-再灌注后神经元的凋亡率、坏死率。用Western bolt检测大脑皮质和海马组织中低氧诱导因子-1α(HIF-1α)、半胱氨酸蛋白酶(caspase-3)、凋亡抑制基因(survivn)蛋白表达量。结果 大鼠局灶性脑缺血-再灌注后大脑皮质和海马区出现神经细胞的坏死和凋亡改变,与假手术组相比HIF-1α、caspase-3蛋白表达增加,分别为HIF-1α0.84±0.03、caspase-3 0.57±0.04、假手术组分别为0.46±0.02、0.17±0.01(P<0.05)、survivn蛋白表达减少0.19±0.02,假手术组为0.31±0.01(P<0.05);给予异丙酚预处理后上述变化显著减轻,HIF-1α0.68±0.02、caspase-3 0.29±0.03、survi...     

Gene therapy for the prevention of ischemia/reperfusion injury in organ transplantation

Introduction of gene therapy into molecular medicine has been gaining increasing interest. Although treatment of various diseases e.g. monogenetic defects or cancer by using gene transfer technologies has been extensively probed, the clinical success has been limited. However, recent experimental data suggest that gene therapy may represent an attractive and powerful approach in preventing ischemia/reperfusion injury as well as organ rejection in transplant recipients. Easy and selective access to the donor organ facilitates the reduction of potentially harmful systemic side effects of gene therapy vectors. By introducing anti-apoptotic or cytoprotective genes, these studies focused on the protection of the transplant from the apoptotic cell death. In addition, down-regulation of adhesion molecules and/or blockade of gene expression in the graft itself also ameliorated ischemia/reperfusion injury. This review summarizes the current progress on gene therapy application in combating ischemia-reperfusion injury in organ transplantation. Although the use of viral vectors is emphasized, non-viral gene transfer techniques are also discussed. Future development of novel, low-immunogenic vectors should further contribute to the minimization of ischemia/reperfusion injury, and thus to the overall success of organ transplantation.     

The bed nucleus of the stria terminalis is critically involved in enhancing associative learning after stressful experience

Exposure to an acute stressful event enhances trace eyeblink conditioning in male rats, even when rats begin training days after the stressor (Shors, 2001). The authors examined whether the bed nucleus of the stria terminalis (BNST), an area involved in stress and anxiety, is critically involved in this effect and, if so, when. The authors found that excitotoxic lesions to the BNST prevented the enhanced conditioning after stressor exposure. In addition, temporary inactivation of the BNST during the stressor did not alter enhanced responding, whereas inactivation during training prevented the enhancement. These data indicate that stressful experience induces persistent changes in the BNST that are necessary for enhancing learning well after the stressful event has ceased.     

Studies on the oxygen radical mechanism involved in the small intestinal reperfusion damage

Characteristic mucosal lesions develop in the small intestine during ischaemia and hypotension. This tissue damage can be further aggravated in the immediate reperfusion phase, presumably secondary to the generation of oxygen free radicals which have been proposed to be generated in this situation through the hypoxanthine-xanthine oxidase system. This was further investigated in the cat small intestine using a standardized regional intestinal hypotension model in which the effects of allopurinol (a xanthine oxidase inhibitor) were compared to those of an exogenous supply of inosine. The grade of mucosal damage, the nucleotide levels, the concentrations of hypoxanthine, total and oxidized glutathione, and of conjugated dienes were measured in the intestinal tissue. The results indicate that oxygen radicals generated by xanthine oxidase are very important, but not the only significant factor in the small intestinal reperfusion damage.     

Lung inflammation is induced by renal ischemia and reperfusion injury as part of the systemic inflammatory syndrome

Introduction

Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs.

Aim

The objective was to study the pulmonary inflammatory systemic response after renal IRI.

Methods

Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE₂ concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR.

Results

Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 ± 0.16 vs. 0.43 ± 0.07, p < 0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 × 10⁴ ± 15.63 vs. 18.1×10⁴ ± 10.5, p < 0.05) 24 h (124 × 10⁴ ± 8.94 vs. 23.2×10⁴ ± 3.5, p < 0.05) and 48 h (79 × 10⁴ ± 15.72 vs. 22.2 × 10⁴ ± 4.2, p < 0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-α, IL-1β, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-α, IL-1β and MCP-1 and BALF PGE₂ concentrations were increased 24 h after renal IRI.

Conclusion

Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal injury.     

晚期缺血预适应促进低氧诱导因子的表达减轻肾脏缺血再灌注损伤

目的: 探讨晚期缺血预适应对肾脏缺血再灌注损伤的作用,以及低氧诱导因子1α(HIF-1α)在其中的作用。方法: 将雄性C57/BL6N小鼠随机分为3组:假手术组(sham)、缺血再灌注组(IR)和缺血预适应组(IPC)。采用夹闭双侧肾蒂30 min后恢复灌注的方法建立肾缺血再灌注小鼠模型;缺血预适应组4 d前给予肾脏15 min预缺血。观察缺血预处理对再灌注后不同时点血肌酐(Scr)、尿素氮(BUN)、肾组织形态学和细胞凋亡的影响。采用免疫组化及Western印迹法,分析HIF-1α在肾组织的表达;采用实时定量RT-PCR法,检测血管内皮生长因子(VEGF)和葡萄糖转运子-1(Glut-1)的mRNA表达。结果: 再灌注24 h后,IPC组小管间质损伤程度较IR组显著减轻,Scr、BUN水平以及小管上皮细胞凋亡明显下降。IPC组的HIF-1α核内表达显著高于IR组,且HIF-1下游靶基因VEGF和Glut-1的mRNA表达亦显著增加。结论: 晚期缺血预适应能够显著改善缺血再灌注后肾脏的形态和功能,这种保护作用可能与促进低氧诱导因子高表达有关。     

Noninvasive evaluation of renal pH homeostasis after ischemia reperfusion injury by CEST‐MRI

Acute kidney injury (AKI) in mice caused by sustained ischemia followed by reperfusion is associated with acute tubular necrosis and renal dysfunctional blood flow. Although the principal role of the kidney is the maintenance of acid–base balance, current imaging approaches are unable to assess this important parameter, and clinical biomarkers are not robust enough in evaluating the severity of kidney damage. Therefore, novel noninvasive imaging approaches are needed to assess the acid–base homeostasis in vivo. This study investigates the usefulness of MRI‐chemical exchange saturation transfer (CEST) pH imaging (through iopamidol injection) in characterizing moderate and severe AKI in mice following unilateral ischemia reperfusion injury. Moderate (20 min) and severe (40 min) ischemia were induced in Balb/C mice, which were imaged at several time points thereafter (Days 0, 1, 2, 7). A significant increase of renal pH values was observed as early as one day after the ischemia reperfusion damage for both moderate and severe ischemia. MRI‐CEST pH imaging distinguished the evolution of moderate from severe AKI. A recovery of normal renal pH values was observed for moderate AKI, whereas a persisting renal pH increase was observed for severe AKI on Day 7. Renal filtration fraction was significantly lower for clamped kidneys (0.54–0.57) in comparison to contralateral kidneys (0.84–0.86) following impairment of glomerular filtration. The severe AKI group showed a reduced filtration fraction even after 7 days (0.38 for the clamped kidneys). Notably, renal pH values were significantly correlated with the histopathological score. In conclusion, MRI‐CEST pH mapping is a valid tool for the noninvasive evaluation of both acid–base balance and renal filtration in patients with ischemia reperfusion injury.     

异丙酚和氯胺酮减轻大鼠肾缺血再灌注损伤

目的:观察异丙酚(propofol)或氯胺酮(ketamine)对缺血再灌注肾损伤(IR)大鼠肾组织病理变化的影响。方法:48只成年Wistar大鼠随机均分成6组,每组8只,分别为假手术组(S组)、缺血再灌注模型组(R组)、小剂量异丙酚(5mg/kg)组(P1组)、大剂量异丙酚(10mg/kg)组(P2组)、小剂量氯胺酮(10mg/kg)组(K1组)、大剂量氯胺酮(20mg/kg)组(K2组)。输液后72h处死所有大鼠,在实验开始及处死大鼠前取4mL静脉血放入低温冰箱中保存待测。处死大鼠后取部分肾脏组织置于甲醛及戊二醛溶液,用于石蜡切片,光镜及电镜观察肾脏组织结构的变化。另取一部分肾组织制作成肾组织匀浆待测。待测指标包括血肌酐(Scr)、血尿素氮(BUN)、丙二醛(MDA)、超氧化物歧化酶(SOD)。结果:与缺血再灌注模型组相比,异丙酚和氯胺酮能显著降低缺血再灌注肾损伤大鼠肾组织中MDA含量(P0.05),增强SOD活性(P0.05),使缺血再灌注肾损伤大鼠肾功能及组织学改变得到明显改善。结论:异丙酚或氯胺酮对肾缺血再灌注损伤有明显拮抗作用,其作用机制可能与药物抗氧化及抗炎作用有关。     

Regeneration of the renal brush border after renal ischemia in rats

An early change following mild renal ischemia is the loss of the renal microvilli, which then regenerate morphologically within 6 h. We studied microvillar regeneration in rats with 25 min of renal artery occlusion and subsequent reflow. At subsequent intervals the rats were injected intraperitoneally with [14C]choline and [3H]leucine; 25 min later they were killed and their renal brush border membranes isolated. At 30 min of reflow of blood there was a 77% reduction in the incorporation of [3H]leucine into microvillar protein compared with that of the opposite control kidney (P less than 0.02). The incorporation rose to normal within 60 min. At 30 min of reflow, the incorporation of [14C]choline into phospholipids increased twofold (P less than 0.005), then returned toward normal values after 2 h. The altered incorporation of tracers was not due to change in membrane turnover or substrate pools. The activities of alkaline phosphatase, gamma-glutamyl transpeptidase, and alpha-glucosidase decreased 50% following ischemia (P less than 0.02) and returned to control values within 2 h. Thus, renal damage severe enough to partly efface microvilli is repaired metabolically within several hours.     

小鼠急性缺血-再灌注肾损伤模型的建立及体会

目的观察应用小鼠制备急性缺血-再灌注性肾损伤模型的效果。方法应用微型动脉夹夹闭小鼠双侧肾动脉制备急性缺血-再灌注肾损伤模型,其中两组分别于术后24h和48h后处死观察肾功能及肾脏病理变化,另一组观察其病情及存活情况14天。结果各次造模成功率均达85%以上;术后24h及48h实验组血清肌酐(Scr)和血尿素氮(BUN)水平明显升高,与对照组比较差异有统计学意义(P<0.01);实验组肾脏外观出现典型"大白肾"表现,镜下出现典型急性肾小管坏死表现,并有较多炎症细胞浸润,肾小管组织学评分与对照组比较差异有统计学意义(P均<0.01);实验组在观察期间逐渐出现典型急肾衰竭表现,至14天末,死亡率达91.7%,而对照组全部正常存活。结论应用微型动脉夹夹闭小鼠双侧肾动脉可制备稳定急性缺血-再灌注肾损伤模型,而且成功率较高。     

他克莫司对大鼠肾缺血再灌注损伤的影响及其机制的研究

目的: 探讨他克莫司对大鼠肾缺血再灌注(I/R)损伤的保护作用及机制。方法: 将60只健康雄性Wistar大鼠随机分为3组:假手术组、I/R组和他克莫司处理组,每组各4个时点( 0.5 h、2 h、6 h、24 h)。建立肾I/R损伤模型;检测各组大鼠血清肌酐(Cr)、肿瘤坏死因子(TNF-α)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;用光学显微镜观察各组大鼠肾组织病理变化;用免疫组化法检测Fas和caspase-3蛋白的表达。结果: 在相应再灌注各时点,他克莫司处理组血清Cr、TNF-α和MDA水平均低于I/R组(P<0.05),而他克莫司处理组血清SOD活性高于I/R组(P<0.05)。他克莫司处理组肾组织损伤程度明显轻于I/R组。与假手术组比较,I/R组凋亡蛋白Fas和caspase-3表达水平显著增加(P<0.05),而他克莫司处理组凋亡蛋白Fas和caspase-3表达水平则低于I/R组(P<0.05)。结论: 他克莫司能有效抑制I/R引起的自由基产生、肾小管上皮细胞凋亡以及TNF-α水平降低,表明他克莫司对大鼠肾I/R损伤具有保护作用。     

Effects of ischemic preconditioning on the systemic and renal hemodynamic changes in renal ischemia reperfusion injury

Background: Ischemic preconditioning (IPC) could protect against subsequent renal ischemia reperfusion injury (IRI). However, the mechanisms underlying IPC remain far from complete. Hence, we explored the effects of IPC on the renal and systemic hemodynamic changes, renal function and morphology, as well the involvement of endothelial and inducible nitric oxide synthase (eNOS/iNOS), and nitric oxide (NO). Methods: Male Sprague-Dawley rats were randomly divided into five groups after right-side nephrectomy: Sham group (surgery without vascular clamping); IRI group (the left renal artery was clamped for 45 min); IPC group (pretreated with 15 min of ischemia and 10 min of reperfusion); IPC + vehicle group (administrated with 0.9% saline 5 min before IPC); and IPC + N^G-nitro-L-arginine methylester (L-NAME) group (pretreated with L-NAME 5 min prior to IPC). The renal and systemic hemodynamic parameters, renal function and morphology, as well as eNOS, iNOS, and NO expression levels in the kidneys were measured at the indicated time points after reperfusion. Results: IPC rats exhibited significant improvements in renal function, morphology, and renal artery blood flow (RABF), without obvious influence on the systemic hemodynamics and renal vein blood flow. Increased eNOS, iNOS, and NO expression levels were detected in the kidneys of IPC rats 24 h after reperfusion. Furthermore, the beneficial effects were fully abolished by the administration of L-NAME. Conclusions: The results suggest that IPC contributes to early restoration of RABF, probably through eNOS/iNOS-mediated NO production, thereby alleviating the renal dysfunction and histological damage caused by IRI.